# Patent

## Title
Highly conserved genes and their use to generate probes and primers for detection of microorganisms

## Abstract

Four highly conserved genes, encoding translation elongation factor Tu, translation elongation factor G, the catalytic subunit of proton-translocating ATPase and the RecA recombinase, are used to generate species-specific, genus-specific, family-specific, group-specific and universal nucleic acid probes and amplification primers to rapidly detect and identify algal, archaeal, bacterial, fungal and parasitical pathogens from clinical specimens for diagnosis. The detection of associated antimicrobial agents resistance and toxin genes are also under the scope of the present invention.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of application Ser. No. 10/089,177, filed Mar. 27, 2002 now abandoned, which is the U.S. national phase under 35 U.S.C. §371 of prior PCT International Application No. PCT/CA00/01150, filed Sep. 28, 2000, which claims the benefit of Canada Application No. 2307010 filed May 19, 2000, and Canada Application No. 2283458, filed Sep. 28, 1999.


SEQUENCE LISTING
Sequence Listing in electronic format entitled GENOM.048NPC.TXT was created on Dec. 29, 2009 and is 1.98 MB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Classical Methods for the Identification of Microorganisms
Microorganisms are classically identified by their ability to utilize different substrates as a source of carbon and nitrogen through the use of biochemical tests such as the API20ETM system (bioMérieux). For susceptibility testing, clinical microbiology laboratories use methods including disk diffusion, agar dilution and broth microdilution. Although identifications based on biochemical tests and antibacterial susceptibility tests are cost-effective, generally two days are required to obtain preliminary results due to the necessity of two successive overnight incubations to identify the bacteria from clinical specimens as well as to determine their susceptibility to antimicrobial agents. There are some commercially available automated systems (i.e. the MicroScanTM system from Dade Behring and the VitekTM system from bioMérieux) which use sophisticated and expensive apparatus for faster microbial identification and susceptibility testing (Stager and Davis, 1992, Clin. Microbiol. Rev. 5:302-327). These systems require shorter incubation periods, thereby allowing most bacterial identifications and susceptibility testing to be performed in less than 6 hours. Nevertheless, these faster systems always require the primary isolation of the bacteria or fingi as a pure culture, a process which takes at least 18 hours for a pure culture or 2 days for a mixed culture. So, the shortest time from sample reception to identification of the pathogen is around 24 hours. Moreover, fingi other than yeasts are often difficult or very slow to grow from clinical specimens. Identification must rely on labor-intensive techniques such as direct microscopic examination of the specimens and by direct and/or indirect immunological assays. Cultivation of most parasites is impractical in the clinical laboratory. Hence, microscopic examination of the specimen, a few immunological tests and clinical symptoms are often the only methods used for an identification that frequently remains presumptive.
The fastest bacterial identification system, the autoSCAN-Walk-AwayTM system (Dade Behring) identifies both gram-negative and gram-positive bacterial species from standardized inoculum in as little as 2 hours and gives susceptibility patterns to most antibiotics in 5 to 6 hours. However, this system has a particularly high percentage (i.e. 3.3 to 40.5%) of non-conclusive identifications with bacterial species other than Enterobacteriaceae (Croizé J., 1995, Lett. Infectiol. 10:109-113; York et al., 1992, J. Clin. Microbiol. 30:2903-2910). For Enterobacteriaceae, the percentage of non-conclusive identifications was 2.7 to 11.4%. The list of microorganisms identified by commercial systems based on classical identification methods is given in Table 15.
A wide variety of bacteria and fungi are routinely isolated and identified from clinical specimens in microbiology laboratories. Tables 1 and 2 give the incidence for the most commonly isolated bacterial and fungal pathogens from various types of clinical specimens. These pathogens are the main organisms associated with nosocomial and community-acquired human infections and are therefore considered the most clinically important.
Clinical Specimens Tested in Clinical Microbiology Laboratories
Most clinical specimens received in clinical microbiology laboratories are urine and blood samples. At the microbiology laboratory of the Centre Hospitalier de l'Université Laval (CHUL), urine and blood account for approximately 55% and 30% of the specimens received, respectively (Table 3). The remaining 15% of clinical specimens comprise various biological fluids including sputum, pus, cerebrospinal fluid, synovial fluid, and others (Table 3). Infections of the urinary tract, the respiratory tract and the bloodstream are usually of bacterial etiology and require antimicrobial therapy. In fact, all clinical samples received in the clinical microbiology laboratory are tested routinely for the identification of bacteria and antibiotic susceptibility.
Conventional Pathogen Identification from Clinical Specimens
Urine Specimens
The search for pathogens in urine specimens is so preponderant in the routine microbiology laboratory that a myriad of tests have been developed. However, the gold standard remains the classical semi-quantitative plate culture method in which 1 μL of urine is streaked on agar plates and incubated for 18-24 hours. Colonies are then counted to determine the total number of colony forming units (CFU) per liter of urine. A bacterial urinary tract infection (UTI) is normally associated with a bacterial count of 107 CFU/L or more in urine. However, infections with less than 107 CFU/L in urine are possible, particularly in patients with a high incidence of diseases or those catheterized (Stark and Maki, 1984, N. Engl. J. Med. 311:560-564). Importantly, approximately 80% of urine specimens tested in clinical microbiology laboratories are considered negative (i.e. bacterial count of less than 107 CFU/L; Table 3). Urine specimens found positive by culture are further characterized using standard biochemical tests to identify the bacterial pathogen and are also tested for susceptibility to antibiotics. The biochemical and susceptibility testing normally require 18-24 hours of incubation.
Accurate and rapid urine screening methods for bacterial pathogens would allow a faster identification of negative specimens and a more efficient treatment and care management of patients. Several rapid identification methods (UriscreenTM, UTIscreenTM, Flash TrackTM DNA probes and others) have been compared to slower standard biochemical methods, which are based on culture of the bacterial pathogens. Although much faster, these rapid tests showed low sensitivities and poor specificities as well as a high number of false negative and false positive results (Koening et al., 1992, J. Clin. Microbiol. 30:342-345; Pezzlo et al., 1992, J. Clin. Microbiol. 30:640-684).
Blood Specimens
The Blood Specimens Received In The Microbiology Laboratory Are Always Submitted For Culture. Blood Culture Systems May Be Manual, Semi-Automated Or Completely Automated. The BACTECTM System (From Becton Dickinson) And The BactalertTM System (From Organon Teklika Corporation) Are The Two Most Widely Used Automated Blood Culture Systems. These Systems Incubate Blood Culture Bottles Under Optimal Conditions For Growth Of Most Bacteria. Bacterial Growth Is Monitored Continuously To Detect Early Positives By Using Highly Sensitive Bacterial Growth Detectors. Once Growth Is Detected, A Gram Stain Is Performed Directly From The Blood Culture And Then Used To Inoculate Nutrient Agar Plates. Subsequently, Bacterial Identification And Susceptibility Testing Are Carried Out From Isolated Bacterial Colonies With Automated Systems As Described Previously. Blood Culture Bottles Are Normally Reported As Negative If No Growth Is Detected After An Incubation Of 6 To 7 Days. Normally, The Vast Majority Of Blood Cultures Are Reported Negative. For Example, The Percentage Of Negative Blood Cultures At The Microbiology Laboratory Of The CHUL For The Period February 1994-January 1995 Was 93.1% (Table 3).
Other Clinical Samples
Upon receipt by the clinical microbiology laboratory, all body fluids other than blood and urine that are from normally sterile sites (i.e. cerebrospinal, synovial, pleural, pericardial and others) are processed for direct microscopic examination and subsequent culture. Again, most clinical samples are negative for culture (Table 3). In all these normally sterile sites, tests for the universal detection of algae, archaea, bacteria, fungi and parasites would be very useful.
Regarding clinical specimens which are not from sterile sites such as sputum or stool specimens, the laboratory diagnosis by culture is more problematic because of the contamination by the normal flora. The bacterial or fungal pathogens potentially associated with the infection are grown and separated from the colonizing microbes using selective methods and then identified as described previously. Of course, the DNA-based universal detection of bacteria would not be useful for the diagnosis of bacterial infections at these non-sterile sites. On the other hand, DNA-based assays for species or genus or family or group detection and identification as well as for the detection of antimicrobial agents resistance genes from these specimens would be very useful and would offer several advantages over classical identification and susceptibility testing methods.
DNA-Based Assays with any Specimen
There is an obvious need for rapid and accurate diagnostic tests for the detection and identification of algae, archaea, bacteria, fingi and parasites directly from clinical specimens. DNA-based technologies are rapid and accurate and offer a great potential to improve the diagnosis of infectious diseases (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.; Bergeron and Ouellette, 1995, Infection 23:69-72; Bergeron and Ouellette, 1998, J Clin Microbiol. 36:2169-72). The DNA probes and amplification primers which are objects of the present invention are applicable for the detection and identification of algae, archaea, bacteria, fingi, and parasites directly from any clinical specimen such as blood, urine, sputum, cerebrospinal fluid, pus, genital and gastro-intestinal tracts, skin or any other type of specimens (Table 3). These assays are also applicable to detection from microbial cultures (e.g. blood cultures, bacterial or fungal colonies on nutrient agar, or liquid cell cultures in nutrient broth). The DNA-based tests proposed in this invention are superior in terms of both rapidity and accuracy to standard biochemical methods currently used for routine diagnosis from any clinical specimens in microbiology laboratories. Since these tests can be performed in one hour or less, they provide the clinician with new diagnostic tools which should contribute to a better management of patients with infectious diseases. Specimens from sources other than humans (e.g. other primates, birds, plants, mammals, farm animals, livestock, food products, environment such as water or soil, and others) may also be tested with these assays.
A High Percentage of Culture-Negative Specimens
Among all the clinical specimens received for routine diagnosis, approximately 80% of urine specimens and even more (around 95%) for other types of normally sterile clinical specimens are negative for the presence of bacterial pathogens (Table 3). It would also be desirable, in addition to identify bacteria at the species or genus or family or group level in a given specimen, to screen out the high proportion of negative clinical specimens with a DNA-based test detecting the presence of any bacterium (i.e. universal bacterial detection). As disclosed in the present invention, such a screening test may be based on DNA amplification by PCR of a highly conserved genetic target found in all bacteria. Specimens negative for bacteria would not be amplified by this assay. On the other hand, those that are positive for any bacterium would give a positive amplification signal. Similarly, highly conserved genes of fungi and parasites could serve not only to identify particular species or genus or family or group but also to detect the presence of any fungi or parasite in the specimen.
Towards the Development of Rapid DNA-Based Diagnostic Tests
A rapid diagnostic test should have a significant impact on the management of infections. DNA probe and DNA amplification technologies offer several advantages over conventional methods for the identification of pathogens and antimicrobial agents resistance genes from clinical samples (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-based Diagnostics in Infectious Disease, Blackwell Scientific Publications, Boston, Mass.). There is no need for culture of the pathogens, hence the organisms can be detected directly from clinical samples, thereby reducing the time associated with the isolation and identification of pathogens. Furthermore, DNA-based assays are more accurate for microbial identification than currently used phenotypic identification systems which are based on biochemical tests and/or microscopic examination. Commercially available DNA-based technologies are currently used in clinical microbiology laboratories, mainly for the detection and identification of fastidious bacterial pathogens such as Mycobacterium tuberculosis, Chlamydia trachomatis, Neisseria gonorrhoeae as well as for the detection of a variety of viruses (Tang Y. and Persing D. H., Molecular detection and identification of microorganisms, In: P. Murray et al., 1999, Manual of Clinical Microbiology, ASM press, 7th edition, Washington D.C.). There are also other commercially available DNA-based assays which are used for culture confirmation assays.
Others have developed DNA-based tests for the detection and identification of bacterial pathogens which are objects of the present invention, for example: Staphylococcus sp. (U.S. Pat. No. 5,437,978), Neisseria sp. (U.S. Pat. No. 5,162,199 and European patent serial no. 0,337,896,131) and Listeria monocytogenes (U.S. Pat. Nos. 5,389,513 and 5,089,386). However, the diagnostic tests described in these patents are based either on rRNA genes or on genetic targets different from those described in the present invention. To our knowledge there are only four patents published by others mentioning the use of any of the four highly conserved gene targets described in the present invention for diagnostic purposes (PCT international publication number WO92/03455 and WO00/14274, European patent publication number 0 133 671 B1, and European patent publication number 0 133 288 A2). WO92/03455 is focused on the inhibition of Candida species for therapeutic purposes. It describes antisense oligonucleotide probes hybridizing to Candida messenger RNA. Two of the numerous mRNA proposed as targets are coding for translation elongation factor 1 (tef1) and the beta subunit of ATPase. DNA amplification or hybrization are not under the scope of their invention and although diagnostic use is briefly mentioned in the body of the application, no specific claim is made regarding diagnostics. WO00/14274 describes the use of bacterial recA gene for identification and speciation of bacteria of the Burkholderia cepacia complex. Specific claims are made on a method for obtaining nucleotide sequence information for the recA gene from the target bacteria and a following comparison with a standard library of nucleotide sequence information, and on the use of PCR for amplification of the recA gene in a sample of interest. However, the use of a discriminatory restriction enzyme in a RFLP procedure is essential to fulfill the speciation and WO00/14274 did not mention that multiple recA probes could be used simultaneously. Patent EP 0 133 288 A2 describes and claims the use of bacterial tuf (and fus) sequence for diagnostics based on hybridization of a tuf (or fus) probe with bacterial DNA. DNA amplification is not under the scope of EP 0 133 288 A2. Nowhere it is mentioned that multiple tuf (or fus) probes could be used simultaneously. No mention is made regarding speciation using tuf (or fus) DNA nucleic acids and/or sequences. The sensitivities of the tuf hybrizations reported are 1×106 bacteria or 1-100 ng of DNA. This is much less sensitive than what is achieved by our assays using nucleic acid amplification technologies.
Although there are phenotypic identification methods which have been used for more than 125 years in clinical microbiology laboratories, these methods do not provide information fast enough to be useful in the initial management of patients. There is a need to increase the speed of the diagnosis of commonly encountered bacterial, fungal and parasitical infections. Besides being much faster, DNA-based diagnostic tests are more accurate than standard biochemical tests presently used for diagnosis because the microbial genotype (e.g. DNA level) is more stable than the phenotype (e.g. physiologic level).
Bacteria, fungi and parasites encompass numerous well-known microbial pathogens. Other microorganisms could also be pathogens or associated with human diseases. For example, achlorophylious algae of the Prototheca genus can infect humans. Archae, especially methanogens, are present in the gut flora of humans (Reeve, J. H., 1999, J. Bacteriol. 181:3613-3617). However, methanogens have been associated to pathologic manifestations in the colon, vagina, and mouth (Belay et al., 1988, Appl. Enviro. Microbiol. 54:600-603; Belay et al., 1990, J. Clin. Microbiol. 28:1666-1668; Weaver et al., 1986, Gut 27:698-704).
In addition to the identification of the infectious agent, it is often desirable to identify harmful toxins and/or to monitor the sensitivity of the microorganism to antimicrobial agents. As revealed in this invention, genetic identification of the microorganism could be performed simultaneously with toxin and antimicrobial agents resistance genes.
Knowledge of the genomic sequences of algal, archaeal, bacterial, fungal and parasitical species continuously increases as testified by the number of sequences available from public databases such as GenBank. From the sequences readily available from those public databases, there is no indication therefrom as to their potential for diagnostic purposes. For determining good candidates for diagnostic purposes, one could select sequences for DNA-based assays for (i) the species-specific detection and identification of commonly encountered bacterial, fungal and parasitical pathogens, (ii) the genus-specific detection and identification of commonly encountered bacterial, fungal or parasitical pathogens, (iii) the family-specific detection and identification of commonly encountered bacterial, fungal or parasitical pathogens, (iv) the group-specific detection and identification of commonly encountered bacterial, fungal or parasitical pathogens, (v) the universal detection of algal, archaeal, bacterial, fungal or parasitical pathogens, and/or (vi) the specific detection and identification of antimicrobial agents resistance genes, and/or (vii) the specific detection and identification of bacterial toxin genes. All of the above types of DNA-based assays may be performed directly from any type of clinical specimens or from a microbial culture.
In our assigned U.S. Pat. No. 6,001,564 and our WO98/20157 patent publication, we described DNA sequences suitable for (i) the species-specific detection and identification of clinically important bacterial pathogens, (ii) the universal detection of bacteria, and (iii) the detection of antimicrobial agents resistance genes.
The WO98/20157 patent publication describes proprietary tuf DNA sequences as well as tuf sequences selected from public databases (in both cases, fragments of at least 100 base pairs), as well as oligonucleotide probes and amplification primers derived from these sequences. All the nucleic acid sequences described in that patent publication can enter in the composition of diagnostic kits or products and methods capable of a) detecting the presence of bacteria and fingi b) detecting specifically at the species, genus, family or group levels, the presence of bacteria and fungi and antimicrobial agents resistance genes associated with these pathogens. However, these methods and kits need to be improved, since the ideal kit and method should be capable of diagnosing close to 100% of microbial pathogens and associated antimicrobial agents resistance genes and toxins genes. For example, infections caused by Enterococcus faecium have become a clinical problem because of its resistance to many antibiotics. Both the detection of these bacteria and the evaluation of their resistance profiles are desirable. Besides that, novel DNA sequences (probes and primers) capable of recognizing the same and other microbial pathogens or the same and additional antimicrobial agents resistance genes are also desirable to aim at detecting more target genes and complement our earlier patent applications.
The present invention improves the assigned application by disclosing new proprietary tuf nucleic acids and/or sequences as well as describing new ways to obtain tuf nucleic acids and/or sequences. In addition we disclose new proprietary atpD and recA nucleic acids and/or sequences. In addition, new uses of tuf, atpD and recA DNA nucleic acids and/or sequences selected from public databases (Table 11) are disclosed.
Highly Conserved Genes for Identification and Diagnostics
Highly conserved genes are useful for identification of microorganisms. For bacteria, the most studied genes for identification of microorganisms are the universally conserved ribosomal RNA genes (rRNA). Among those, the principal targets used for identification purposes are the small subunit (SSU) ribosomal 16S rRNA genes (in prokaryotes) and 18S rRNA genes (in eukaryotes) (Relman and Persing, Genotyping Methods for Microbial Identification, In: D. H. Persing, 1996, PCR Protocols for Emerging Infectious Diseases, ASM Press, Washington D.C.). The rRNA genes are also the most commonly used targets for universal detection of bacteria (Chen et al., 1988, FEMS Microbiol. Lett. 57:19-24; McCabe et al., 1999, Mol. Genet. Metabol. 66:205-211) and fungi (Van Burik et al., 1998, J. Clin. Microbiol. 36:1169-1175).
However, it may be difficult to discriminate between closely related species when using primers derived from the 16S rRNA. In some instances, 16S rRNA sequence identity may not be sufficient to guarantee species identity (Fox et al., 1992, Int. J. Syst. Bacteriol. 42:166-170) and it has been shown that inter-operon sequence variation as well as strain to strain variation could undermine the application of 16S rRNA for identification purposes (Clayton et al., 1995, Int. J. Syst. Bacteriol. 45:595-599). The heat shock proteins (HSP) are another family of very conserved proteins. These ubiquitous proteins in bacteria and eukaryotes are expressed in answer to external stress agents. One of the most described of these HSP is HSP 60. This protein is very conserved at the amino acid level, hence it has been useful for phylogenetic studies. Similar to 16S rRNA, it would be difficult to discriminate between species using the HSP 60 nucleotide sequences as a diagnostic tool. However, Goh et al. identified a highly conserved region flanking a variable region in HSP 60, which led to the design of universal primers amplifying this variable region (Goh et al., U.S. Pat. No. 5,708,160). The sequence variations in the resulting amplicons were found useful for the design of species-specific assays.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a specific, ubiquitous and sensitive method using probes and/or amplification primers for determining the presence and/or amount of nucleic acids:

    
    
        from any algal, archaeal, bacterial, fungal or parasitical species in any sample suspected of containing said nucleic acids, and optionally,
        from specific microbial species or genera selected from the group consisting of the species or genera listed in Table 4, and optionally,
        from an antimicrobial agents resistance gene selected from the group consisting of the genes listed in Table 5, and optionally,
        from a toxin gene selected from the group consisting of the genes listed in Table 6,
        
            wherein each of said nucleic acids or a variant or part thereof comprises a selected target region hybridizable with said probes or primers;
            said method comprising the steps of contacting said sample with said probes or primers and detecting the presence and/or amount of hybridized probes or amplified products as an indication of the presence and/or amount of said any microbial species, specific microbial species or genus or family or group and antimicrobial agents resistance gene and/or toxin gene.
        
        
    
    


In a specific embodiment, a similar method directed to each specific microbial species or genus or family or group detection and identification, antimicrobial agents resistance genes detection, toxin genes detection, and universal bacterial detection, separately, is provided.
In a more specific embodiment, the method makes use of DNA fragments from conserved genes (proprietary sequences and sequences obtained from public databases), selected for their capacity to sensitively, specifically and ubiquitously detect the targeted algal, archaeal, bacterial, fungal or parasitical nucleic acids.
In a particularly preferred embodiment, oligonucleotides of at least 12 nucleotides in length have been derived from the longer DNA fragments, and are used in the present method as probes or amplification primers. To be a good diagnostic candidate, an oligonucleotide of at least 12 nucleotides should be capable of hybridizing with nucleic acids from given microorganism(s), and with substantially all strains and representatives of said microorganism(s); said oligonucleotide being species-, or genus-, or family-, or group-specific or universal.
In another particularly preferred embodiment, oligonucleotides primers and probes of at least 12 nucleotides in length are designed for their specificity and ubiquity based upon analysis of our databases of tuf, atpD and recA sequences. These databases are generated using both proprietary and public sequence information. Altogether, these databases form a sequence repertory useful for the design of primers and probes for the detection and identification of algal, archaeal, bacterial, fungal and parasitical microorganisms. The repertory can also be subdivided into subrepertories for sequence analysis leading to the design of various primers and probes.
The tuf, atpD and recA sequences databases as a product to assist the design of oligonucleotides primers and probes for the detection and identification of algal, archaeal, bacterial, fungal and parasitical microorganisms are also covered.
The proprietary oligonucleotides (probes and primers) are also another object of this invention.
Diagnostic kits comprising probes or amplification primers such as those for the detection of a microbial species or genus or family or phylum or group selected from the following list consisting of Abiotrophia adiacens, Acinetobacter baumanii, Actinomycetae, Bacteroides, Cytophaga and Flexibacter phylum, Bacteroides fragilis, Bordetella pertussis, Bordetella sp., Campylobacter jejuni and C. coli, Candida albicans, Candida dubliniensis, Candida glabrata, Candida guillierinondii, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Candida sp., Chlamydia pneumoniae, Chlamydia trachomatis, Clostridium sp., Corynebacterium sp., Crypococcus neoformans, Cryptococcus sp., Cryptosporidium parvum, Entamoeba sp., Enterobacteriaceae group, Enterococcus casseliflavus-flavescens-gallinarum group, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus sp., Escherichia coli and Shigella sp. group, Gemella sp., Giardia sp., Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Legionella sp., Leishmania sp., Mycobacteriaceae family, Mycoplasma pneumoniae, Neisseria gonorrhoeae, platelets contaminants group (see Table 14), Pseudomonas aeruginosa, Pseudomonads group, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus sp., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sp., Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma sp., Trypanosomatidae family, are also objects of the present invention.
Diagnostic kits further comprising probes or amplification primers for the detection of an antimicrobial agents resistance gene selected from the group listed in Table 5 are also objects of this invention.
Diagnostic kits further comprising probes or amplification primers for the detection of a toxin gene selected from the group listed in Table 6 are also objects of this invention.
Diagnostic kits further comprising probes or amplification primers for the detection of any other algal, archaeal, bacterial, fungal or parasitical species than those specifically listed herein, comprising or not comprising those for the detection of the specific microbial species or genus or family or group listed above, and further comprising or not comprising probes and primers for the antimicrobial agents resistance genes listed in Table 5, and further comprising or not comprising probes and primers for the toxin genes listed in Table 6 are also objects of this invention.
In a preferred embodiment, such a kit allows for the separate or the simultaneous detection and identification of the above-listed microbial species or genus or family or group; or universal detection of algae, archaea, bacteria, fungi or parasites; or antimicrobial agents resistance genes; or toxin genes; or for the detection of any microorganism (algae, archaea, bacteria, fungi or parasites).
In the above methods and kits, probes and primers are not limited to nucleic acids and may include, but are not restricted to analogs of nucleotides such as: inosine, 3-nitropyrrole nucleosides (Nichols et al., 1994, Nature 369:492-493), Linked Nucleic Acids (LNA) (Koskin et al., 1998, Tetrahedron 54:3607-3630), and Peptide Nucleic Acids (PNA) (Egholm et al., 1993, Nature 365:566-568).
In the above methods and kits, amplification reactions may include but are not restricted to: a) polymerase chain reaction (PCR), b) ligase chain reaction (LCR), c) nucleic acid sequence-based amplification (NASBA), d) self-sustained sequence replication (3SR), e) strand displacement amplification (SDA), f) branched DNA signal amplification (bDNA), g) transcription-mediated amplification (TMA), h) cycling probe technology (CPT), i) nested PCR, j) multiplex PCR, k) solid phase amplification (SPA), l) nuclease dependent signal amplification (NDSA), m) rolling circle amplification technology (RCA), n) Anchored strand displacement amplification, o) Solid-phase (immobilized) rolling circle amplification.
In the above methods and kits, detection of the nucleic acids of target genes may include real-time or post-amplification technologies. These detection technologies can include, but are not limited to, fluorescence resonance energy transfer (FRET)-based methods such as adjacent hybridization to FRET probes (including probe-probe and probe-primer methods), TaqMan, Molecular Beacons, scorpions, nanoparticle probes and Sunrise (Amplifluor). Other detection methods include target genes nucleic acids detection via immunological methods, solid phase hybridization methods on filters, chips or any other solid support, whether the hybridization is monitored by fluorescence, chemiluminescence, potentiometry, mass spectrometry, plasmon resonance, polarimetry, colorimetry, or scanometry. Sequencing, including sequencing by dideoxy termination or sequencing by hybridization, e.g. sequencing using a DNA chip, is another possible method to detect and identify the nucleic acids of target genes.
In a preferred embodiment, a PCR protocol is used for nucleic acid amplification, in diagnostic method as well as in method of construction of a repertory of nucleic acids and deduced sequences.
In a particularly preferred embodiment, a PCR protocol is provided, comprising, an initial denaturation step of 1-3 minutes at 95° C., followed by an amplification cycle including a denaturation step of one second at 95° C. and an annealing step of 30 seconds at 45-65° C., without any time allowed specifically for the elongation step. This PCR protocol has been standardized to be suitable for PCR reactions with most selected primer pairs, which greatly facilitates the testing because each clinical sample can be tested with universal, species-specific, genus-specific, antimicrobial agents resistance gene and toxin gene PCR primers under uniform cycling conditions. Furthermore, various combinations of primer pairs may be used in multiplex PCR assays.
It is also an object of the present invention that tuf, atpD and recA sequences could serve as drug targets and these sequences and means to obtain them revealed in the present invention can assist the screening, design and modeling of these drugs.
It is also an object of the present invention that tuf, atpD and recA sequences could serve for vaccine purposes and these sequences and means to obtain them revealed in the present invention can assist the screening, design and modeling of these vaccines.
We aim at developing a universal DNA-based test or kit to screen out rapidly samples which are free of algal, archaeal, bacterial, fungal or parasitical cells. This test could be used alone or combined with more specific identification tests to detect and identify the above algal and/or archaeal and/or bacterial and/or fungal and/or parasitical species and/or genera and/or family and/or group and to determine rapidly the bacterial resistance to antibiotics and/or presence of bacterial toxins. Although the sequences from the selected antimicrobial agents resistance genes are available from public databases and have been used to develop DNA-based tests for their detection, our approach is unique because it represents a major improvement over current diagnostic methods based on bacterial cultures. Using an amplification method for the simultaneous or independent or sequential microbial detection-identification and antimicrobial resistance genes detection, there is no need for culturing the clinical sample prior to testing. Moreover, a modified PCR protocol has been developed to detect all target DNA sequences in approximately one hour under uniform amplification conditions. This procedure should save lives by optimizing treatment, should diminish antimicrobial agents resistance because less antibiotics will be prescribed, should reduce the use of broad spectrum antibiotics which are expensive, decrease overall health care costs by preventing or shortening hospitalizations, and side effects of drugs, and decrease the time and costs associated with clinical laboratory testing.
In another embodiment, sequence repertories and ways to obtain them for other gene targets are also an object of this invention, such is the case for the hexA nucleic acids and/or sequences of Streptococci.
In yet another embodiment, for the detection of mutations associated with antibiotic resistance genes, we built repertories to distinguish between point mutations reflecting only gene diversity and point mutations involved in resistance. Such repertories and ways to obtain them for pbp1a, pbp2b and pbp2x genes of sensitive and penicillin-resistant Streptoccoccus pneumoniae and also for gyrA and parC gene fragments from various bacterial species are also an object of the present invention.
The diagnostic kits, primers and probes mentioned above can be used to identify algae, archaea, bacteria, fingi, parasites, antimicrobial agents resistance genes and toxin genes on any type of sample, whether said diagnostic kits, primers and probes are used for in vitro or in situ applications. The said samples may include but are not limited to: any clinical sample, any environment sample, any microbial culture, any microbial colony, any tissue, and any cell line.
It is also an object of the present invention that said diagnostic kits, primers and probes can be used alone or in conjunction with any other assay suitable to identify microorganisms, including but not limited to: any immunoassay, any enzymatic assay, any biochemical assay, any lysotypic assay, any serological assay, any differential culture medium, any enrichment culture medium, any selective culture medium, any specific assay medium, any identification culture medium, any enumeration culture medium, any cellular stain, any culture on specific cell lines, and any infectivity assay on animals.
In the methods and kits described herein below, the oligonucleotide probes and amplification primers have been derived from larger sequences (i.e. DNA fragments of at least 100 base pairs). All DNA fragments have been obtained either from proprietary fragments or from public databases. DNA fragments selected from public databases are newly used in a method of detection according to the present invention, since they have been selected for their diagnostic potential.
In another embodiment, the amino acid sequences translated from the repertory of tuf, atpD and recA nucleic acids and/or sequences are also an object of the present invention.
It is clear to the individual skilled in the art that other oligonucleotide sequences appropriate for (i) the universal detection of algae, archaea, bacteria, fungi or parasites, (ii) the detection and identification of the above microbial species or genus or family or group, and (iii) the detection of antimicrobial agents resistance genes, and (iv) the detection of toxin genes, other than those listed in Tables 39-41, 59-60, 70-76, 77-79, and 81-92 may also be derived from the proprietary fragments or selected public database sequences. For example, the oligonucleotide primers or probes may be shorter or longer than the ones chosen; they may also be selected anywhere else in the proprietary DNA fragments or in the sequences selected from public databases; they may be also variants of the same oligonucleotide. If the target DNA or a variant thereof hybridizes to a given oligonucleotide, or if the target DNA or a variant thereof can be amplified by a given oligonucleotide PCR primer pair, the converse is also true; a given target DNA may hybridize to a variant oligonucleotide probe or be amplified by a variant oligonucleotide PCR primer. Alternatively, the oligonucleotides may be designed from any DNA fragment sequences for use in amplification methods other than PCR. Consequently, the core of this invention is the identification of universal, species-specific, genus-specific, family-specific, group-specific, resistance gene-specific, toxin gene-specific genomic or non-genomic DNA fragments which are used as a source of specific and ubiquitous oligonucleotide probes and/or amplification primers. Although the selection and evaluation of oligonucleotides suitable for diagnostic purposes requires much effort, it is quite possible for the individual skilled in the art to derive, from the selected DNA fragments, oligonucleotides other than the ones listed in Tables 39-41, 59-60, 70-76, 77-79, and 81-92 which are suitable for diagnostic purposes. When a proprietary fragment or a public databases sequence is selected for its specificity and ubiquity, it increases the probability that subsets thereof will also be specific and ubiquitous.
Since a high percentage of clinical specimens are negative for bacteria (Table 3), DNA fragments having a high potential for the selection of universal oligonucleotide probes or primers were selected from proprietary and public database sequences. The amplification primers were selected from genes highly conserved in algae, archaea, bacteria, fungi and parasites, and are used to detect the presence of any algal, archaeal, bacterial, fungal or parasitical pathogen in clinical specimens in order to determine rapidly whether it is positive or negative for algae, archaea, bacteria, fungi or parasites. The selected genes, designated tuf, fus, atpD and recA, encode respectively 2 proteins (elongation factors Tu and G) involved in the translational process during protein synthesis, a protein (beta subunit) responsible for the catalytic activity of proton pump ATPase and a protein responsible for the homologous recombination of genetic material. The alignments of tuf, atpD and recA sequences used to derive the universal primers include both proprietary and public database sequences. The universal primer strategy allows the rapid screening of the numerous negative clinical specimens (around 80% of the specimens received, see Table 3) submitted for microbiological testing.
Table 4 provides a list of the archaeal, bacterial, fungal and parasitical species for which tuf and/or atpD and/or recA nucleic acids and/or sequences are revealed in the present invention. Tables 5 and 6 provide a list of antimicrobial agents resistance genes and toxin genes selected for diagnostic purposes. Table 7 provides the origin of tuf atpD and recA nucleic acids and/or sequences listed in the sequence listing. Tables 8-10 and 12-14 provide lists of species used to test the specificity, ubiquity and sensitivity of some assays described in the examples. Table 11 provides a list of microbial species for which tuf and/or atpD and/or recA sequences are available in public databases. Table 15 lists the microorganisms identified by commercial systems. Tables 16-18 are part of Example 42, whereas Tables 19-20 are part of Example 43. Tables 21-22 illustrate Example 44, whereas Tables 23-25 illustrate Example 45.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1 and 2 illustrate the principal subdivisions of the tuf and atpD sequences repertories, respectively. For the design of primers and probes, depending on the needs, one may want to use the complete data set illustrated on the top of the pyramid or use only a subset illustrated by the different branching points. Smaller subdivisions, representing groups, families, genus and species, could even be made to extend to the bottom of the pyramid. Because the tuf and atpD sequences are highly conserved and evolved with each species, the design of primers and probes does not need to include all the sequences within the database or its subdivisions. As illustrated in Annexes IV to XX, XXIII to XXXI, XXXVIII and XLII, depending on the use, sequences from a limited number of species can be carefully selected to represent: i) only the main phylogenetic branches from which the intended probes and primers need to be differentiating, and ii) only the species for which they need to be matching. However, for ubiquity purposes, and especially for primers and probes identifying large groups of species (genus, family, group or universal, or sequencing primers), the more data is included into the sequence analysis, the better the probes and primers will be suitable for each particular intended use. Similarly, for specificity purposes, a larger data set (or repertory) ensures optimal primers and probes design by reducing the chance of employing nonspecific oligonucleotides.
FIG. 3 illustrates the approach used to design specific amplification primers from fusA as well as from the region between the end of fusA and the beginning of tuf in the streptomycin (str) operon (referred to as the fusA-tuf intergenic spacer in Table 7). Shown is a schematic organization of universal amplification primers (SEQ ID NOs. 1221-1229) in the str operon. Amplicon sizes are given in bases pairs. Drawing not to scale, as the fusA-tuf intergenic spacer size varies depending on the bacterial species. Indicated amplicon lengths are for E. coli. 
FIGS. 4 to 6 are illustrations to Example 42, whereas FIGS. 7 to 10 illustrate Example 43. FIGS. 11 and 12 illustrate Example 44.
FIG. 4. Abridged multiple amino acid sequence alignment of the partial tuf gene products from selected species illustrated using the program Alscript. Residues highly conserved in bacteria are boxed in grey and gaps are represented with dots. Residues in reverse print are unique to the enterococcal tufB as well as to streptococcal and lactococcal tuf gene products. Numbering is based on E. coli EF-Tu and secondary structure elements of E. coli EF-Tu are represented by cylinders (α-helices) and arrows (β-strands). The sequences shown correspond to SEQ ID NO's: 2630 to 2667.
FIG. 5. Distance matrix tree of bacterial EF-Tu based on amino acid sequence homology. The tree was constructed by the neighbor-joining method. The tree was rooted using archeal and eukaryotic EF-1α genes as the outgroup. The scale bar represents 5% changes in amino acid sequence, as determined by taking the sum of all of the horizontal lines connecting two species.
FIG. 6. Southern hybridization of BglII/XbaI digested genomic DNAs of some enterococci (except for E. casseliflavus and E. gallinarum whose genomic DNA was digested with BamHI/PvuII) using the tufA gene fragment of E. faecium as probes. The sizes of hybridizing fragments are shown in kilobases. Strains tested are listed in Table 16.
FIG. 7. Pantoea and Tatumella species specific signature indel in atpD genes. The nucleotide positions given are for E. coli atpD sequence (GenBank accession no. V00267). Numbering starts from the first base of the initiation codon.
FIG. 8: Trees based on sequence data from tuf (left side) and atpD (right side). The phylogenetic analysis was performed using the Neighbor-Joining method calculated using the Kimura two-parameter method. The value on each branch indicates the occurrence (%) of the branching order in 750 bootstrapped trees.
FIG. 9: Phylogenetic tree of members of the family Enterobacteriaceae based on tuf (a), atpD (b), and 16S rDNA (c) genes. Trees were generated by neighbor-joining method calculated using the Kimura two-parameter method. The value on each branch is the percentage of bootstrap replications supporting the branch. 750 bootstrap replications were calculated.
FIG. 10: Plot of tuf distances versus 16S rDNA distances (a), atpD distances versus 16S rDNA distances (b), and atpD distances versus tuf distances (c). Symbols: ◯, distances between pairs of strains belonging to the same species; ●, distances between E. coli strains and Shigella strains; □, distances between pairs belonging to the same genus; ▪, distances between pairs belonging to different genera; Δ, distances between pairs belonging to different families.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present inventors reasoned that comparing the published Haemophilus influenzae and Mycoplasma genitalium genomes and searching for conserved genes could provide targets to develop useful diagnostic primers and probes. This sequence comparison is highly informative as these two bacteria are distantly related and most genes present in the minimal genome of M. genitalium are likely to be present in every bacterium. Therefore genes conserved between these two bacteria are likely to be conserved in all other bacteria.
Following the genomic comparison, it was found that several protein-coding genes were conserved in evolution. Highly conserved proteins included the translation elongation factors G (EF-G) and Tu (EF-Tu) and the β subunit of F0F1 type ATP-synthase, and to a lesser extent, the RecA recombinase. These four proteins coding genes were selected amongst the 20 most conserved genes on the basis that they all possess at least two highly conserved regions suitable for the design of universal amplification and sequencing primers. Moreover, within the fragment amplified by these primers, highly conserved and more variable regions are also present hence suggesting it might be possible to rapidly obtain sequence information from various microbial species to design universal as well as species-, genus-, family-, or group-specific primers and probes of potential use for the detection and identification and/or quantification of microorganisms.
Translation elongation factors are members of a family of GTP-binding proteins which intervene in the interactions of tRNA molecules with the ribosome machinery during essential steps of protein synthesis. The role of elongation factor Tu is to facilitate the binding of aminoacylated tRNA molecules to the A site of the ribosome. The eukaryotic, archaeal (archaebacterial) and algal homolog of EF-Tu is called elongation factor 1 alpha (EF-1α). All protein synthesis factors originated from a common ancestor via gene duplications and fusions (Cousineau et al., 1997, J. Mol. Evol. 45:661-670). In particular, elongation factor G (EF-G), although having a functional role in promoting the translocation of aminoacyl-tRNA molecules from the A site to the P site of the ribosome, shares sequence homologies with EF-Tu and is thought to have arisen from the duplication and fusion of an ancestor of the EF-Tu gene.
In addition, EF-Tu is known to be the target for antibiotics belonging to the elfamycin's group as well as to other structural classes (Anborgh and Parmeggiani, 1991, EMBO J. 10:779-784; Luiten et al., 1992, European patent application serial No. EP 0 466 251 A1). EF-G for its part, is the target of the antibiotic fusidic acid. In addition to its crucial activities in translation, EF-Tu has chaperone-like functions in protein folding, protection against heat denaturation of proteins and interactions with unfolded proteins (Caldas et al., 1998, J. Biol. Chem. 273:11478-11482). Interestingly, a form of the EF-Tu protein has been identified as a dominant component of the periplasm of Neisseria gonorrhoeae (Porcella et al., 1996, Microbiology 142:2481-2489), hence suggesting that at least in some bacterial species, EF-Tu might be an antigen with vaccine potential.
F0F1 type ATP-synthase belongs to a superfamily of proton-translocating ATPases divided in three major families: P, V and F (Nelson and Taiz, 1989, TIBS 14:113-116). P-ATPases (or E1-E2 type) operate via a phosphorylated intermediate and are not evolutionarily related to the other two families. V-ATPases (or V0V1 type) are present on the vacuolar and other endomembranes of eukaryotes, on the plasma membrane of archaea (archaebacteria) and algae, and also on the plasma membrane of some eubacteria especially species belonging to the order Spirochaetales as well as to the Chlamydiaceae and Deinococcaceae families. F-ATPases (or F0F1 type) are found on the plasma membrane of most eubacteria, on the inner membrane of mitochondria and on the thylakoid membrane of chloroplasts. They function mainly in ATP synthesis. They are large multimeric enzymes sharing numerous structural and functional features with the V-ATPases. F and V-type ATPases have diverged from a common ancestor in an event preceding the appearance of eukaryotes. The β subunit of the F-ATPases is the catalytic subunit and it possesses low but significant sequence homologies with the catalytic A subunit of V-ATPases.
The translation elongation factors EF-Tu, EF-G and EF-1α and the catalytic subunit of F or V-types ATP-synthase, are highly conserved proteins sometimes used for phylogenetic analysis and their genes are also known to be highly conserved (Iwabe et al., 1989, Proc. Natl. Acad. Sci. USA 86:9355-9359, Gogarten et al., 1989, Proc. Natl. Acad. Sci. USA 86:6661-6665, Ludwig et al., 1993, Antonie van Leeuwenhoek 64:285-305). A recent BLAST (Altschul et al., 1997, J. Mol. Biol. 215:403-410) search performed by the present inventors on the GenBank, European Molecular Biology Laboratory (EMBL), DNA Database of Japan (DDBJ) and specific genome project databases indicated that throughout bacteria, the EF-Tu and the β subunit of F0F1 type ATP-synthase genes may be more conserved than other genes that are well conserved between H. influenzae and M. genitalium. 
The RecA recombinase is a multifunctional protein encoded by the recA gene. It plays a central role in homologous recombination, it is critical for the repair of DNA damage and it is involved in the regulation of the SOS system by promoting the proteolytic digestion of the LexA repressor. It is highly conserved in bacteria and could serve as a useful genetic marker to reconstruct bacterial phylogeny (Miller and Kokjohn, 1990, Annu. Rev. Microbiol. 44:365-394). Although RecA possesses some highly conserved sequence segments that we used to design universal primers aimed at sequencing the recA fragments, it is clearly not as well conserved EF-G, EF-Tu and β subunit of F0F1 type ATP-synthase. Hence, RecA may not be optimal for universal detection of bacteria with high sensitivity but it was chosen because preliminary data indicated that EF-G, EF-Tu and β subunit of F0F1 type ATP-synthase may sometimes be too closely related to find specific primer pairs that could discriminate between certain very closely related species and genera. While RecA, EF-G, EF-Tu and β subunit of F0F1 type ATP-synthase genes, possesses highly conserved regions suitable for the design of universal sequencing primers, the less conserved region between primers should be divergent enough to allow species-specific and genus-specific primers in those cases.
Thus, as targets to design primers and probes for the genetic detection of microorganisms, the present inventors have focused on the genes encoding these four proteins: tuf, the gene for elongation factor Tu (EF-Tu); fus, the gene for the elongation factor G (EF-G); atpD, the gene for β subunit of F0F1 type ATP-synthase; and recA, the gene encoding the RecA recombinase. In several bacterial genomes tuf is often found in two highly similar duplicated copies named tufA and tufB (Filer and Furano, 1981, J. Bacteriol. 148:1006-1011, Sela et al., 1989, J. Bacteriol. 171:581-584). In some particular cases, more divergent copies of the tuf genes can exist in some bacterial species such as some actinomycetes (Luiten et al. European patent application publication No. EP 0 446 251 A1; Vijgenboom et al., 1994, Microbiology 140:983-998) and, as revealed as part of this invention, in several enterococcal species. In several bacterial species, tuf is organized in an operon with its homolog gene for the elongation factor G (EF-G) encoded by the fusA gene (FIG. 3). This operon is often named the str operon. The tuf fus, atpD and recA genes were chosen as they are well conserved in evolution and have highly conserved stretches as well as more variable segments. Moreover, these four genes have eukaryotic orthologs which are described in the present invention as targets to identify fungi and parasites. The eukaryotic homolog of elongation factor Tu is called elongation factor 1-alpha (EF-1α) (gene name: tef, tef1, ef1, ef-1 or EF-1). In fungi, the gene for EF-1α occurs sometimes in two or more highly similar duplicated copies (often named tef1, tef2, tef3 . . . ). In addition, eukaryotes have a copy of elongation factor Tu which is originating from their organelle genome ancestry (gene name: tuf1, tufM or tufA). For the purpose of the current invention, the genes for these four functionally and evolutionarily linked elon-gation factors (bacterial EF-Tu and EF-G, eukaryotic EF-1α, and organellar EF-Tu) will hereafter be designated as <<tuf nucleic acids and/or sequences>>. The eukaryotic (mitochondrial) F0F1 type ATP-synthase beta subunit gene is named atp2 in yeast. For the purpose of the current invention, the genes of catalytic sub-unit of either F or V-type ATP-synthase will hereafter be designated as <<atpD nucleic acids and/or sequences>>. The eukaryotic homologs of RecA are distributed in two families, typified by the Rad51 and Dmc1 proteins. Archaeal homologs of RecA are called RadA. For the purpose of the current invention, the genes corres-ponding to the latter proteins will hereafter be designated as <<recA nucleic acids and/or sequences>>.
In the description of this invention, the terms <<nucleic acids>> and <<sequences>> might be used interchangeably. However, <<nucleic acids>> are chemical entities while <<sequences>> are the pieces of information derived from (inherent to) these <<nucleic acids>>. Both nucleic acids and sequences are equiva-lently valuable sources of information for the matter pertaining to this invention.
Analysis of multiple sequence alignments of tuf and atpD sequences permitted the design of oligonucleotide primers (and probes) capable of amplifying (or hybridizing to) segments of tuf (and/or fus) and atpD genes from a wide variety of bacterial species (see Examples 1 to 4, 24 and 26, and Table 7). Sequencing and amplification primer pairs for tuf nucleic acids and/or sequences are listed in Annex I and hybridization probes are listed in Annexes III and XLVII. Sequencing and amplification primer pairs for atpD nucleic acids and/or sequences are listed in Annex II. Analysis of the main subdivisions of tuf and atpD sequences (see FIGS. 1 and 2) permitted to design sequencing primers amplifying specifically each of these subdivisions. It should be noted that these sequencing primers could also be used as universal primers. However, since some of these sequencing primers include several variable sequence (degenerated) positions, their sensitivity could be lower than that of universal primers developed for diagnostic purposes. Further subdivisions could be done on the basis of the various phyla where these genes are encountered.
Similarly, analysis of multiple sequence alignments of recA sequences present in the public databases permitted the design of oligonucleotide primers capable of amplifying segments of recA genes from a wide variety of bacterial species. Sequencing and amplification primer pairs for recA sequences are listed in Annex XXI. The main subdivisions of recA nucleic acids and/or sequences comprise recA, radA, rad51 and dmc1. Further subdivisions could be done on the basis of the various phyla where these genes are encountered.
The present inventor's strategy is to get as much sequence data information from the four conserved genes (tuf, fus, atpD and recA). This ensemble of sequence data forming a repertory (with subrepertories corresponding to each target gene and their main sequence subdivisions) and then using the sequence information of the sequence repertory (or subrepertories) to design primer pairs that could permit either universal detection of algae or archaea or bacteria or fungi or parasites, detection of a family or group of microorganism (e.g. Enterobacteriaceae), detection of a genus (e.g. Streptococcus) or finally a specific species (e.g. Staphylococcus aureus). It should be noted that for the purpose of the present invention a group of microorganisms is defined depending on the needs of the particular diagnostic test. It does not need to respect a particular taxonomical grouping or phylum. See Example 12 where primers were designed to amplify a group a bacteria consisting of the 17 major bacterial species encountered as contaminants of platelet concentrates. Also remark that in that Example, the primers are not only able to sensitively and rapidly detect at least the 17 important bacterial species, but could also detect other species as well, as shown in Table 14. In these circumstances the primers shown in Example 12 are considered universal for platelet-contaminating bacteria. To develop an assay specific for the latter, one or more primers or probes specific to each species could be designed. Another example of primers and/or probes for group detection is given by the Pseudomonad group primers. These primers were designed based upon alignment of tuf sequences from real Pseudomonas species as well as from former Pseudomonas species such as Stenotrophomonas maltophilia. The resulting primers are able to amplify all Pseudomonas species tested as well as several species belonging to different genera, hence as being specific for a group including Pseudomonas and other species, we defined that group as Pseudomonads, as several members were former Pseudomonas. 
For certain applications, it may be possible to develop a universal, group, family or genus-specific reaction and to proceed to species identification using sequence information within the amplicon to design species-specific internal probes or primers, or alternatively, to proceed directly by sequencing the amplicon. The various strategies will be discussed further below.
The ensembles formed by public and proprietary tuf, atpD and recA nucleic acids and/or sequences are used in a novel fashion so they constitute three databases containing useful information for the identification of microorganisms.
Sequence repertories of other gene targets were also built to solve some specific identification problems especially for microbial species genetically very similar to each other such as E. coli and Shigella (see Example 23). Based on tuf, atpD and recA sequences, Streptococcus pneumoniae is very difficult to differentiate from the closely related species S. oralis and S. mitis. Therefore, we elected to built a sequence repertory from hexA sequences (Example 19), a gene much more variable than our highly conserved tuf, atpD and recA nucleic acids and/or sequences.
For the detection of mutations associated with antibiotic resistance genes, we also built repertories to distinguish between point mutations reflecting only gene diversity and point mutations involved in resistance. This was done for pbp1a, pbp2b and pbp2x genes of penicillin-resistant and sensitive Streptoccoccus pneumoniae (Example 18) and also for gyrA and parC gene fragments of various bacterial species for which quinolone resistance is important to monitor.
Oligonucleotide Primers and Probes Design and Synthesis
The tuf fus, atpD and recA DNA fragments sequenced by us and/or selected from public databases (GenBank and EMBL) were used to design oligonucleotides primers and probes for diagnostic purposes. Multiple sequence alignments were made using subsets of the tuf or atpD or recA sequences repertory. Subsets were chosen to encompass as much as possible of the targetted microorganism(s) DNA sequence data and also include sequence data from phylogenetically related microorganisms from which the targetted microorganism(s) should be distinguished. Regions suitable for primers and probes should be conserved for the targetted microorganism(s) and divergent for the microorganisms from which the targetted microorganism(s) should be distinguished. The large amount of tuf or atpD or recA sequences data in our repertory permits to reduce trial and errors in obtaining specific and ubiquitous primers and probes. We also relied on the corresponding peptide sequences of tuf, fus, atpD and recA nucleic acids and/or sequences to facilitate the identification of regions suitable for primers and probes design. As part of the design rules, all oligonucleotides (probes for hybridization and primers for DNA amplification by PCR) were evaluated for their suitability for hybridization or PCR amplification by computer analysis using standard programs (i.e. the Genetics Computer Group (GCG) programs and the primer analysis software OligoTM 5.0). The potential suitability of the PCR primer pairs was also evaluated prior to the synthesis by verifying the absence of unwanted features such as long stretches of one nucleotide and a high proportion of G or C residues at the 3′ end (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). Oligonucleotide probes and amplification primers were synthesized using an automated DNA synthesizer (Perkin-Elmer Corp., Applied Biosystems Division).
The oligonucleotide sequence of primers or probes may be derived from either strand of the duplex DNA. The primers or probes may consist of the bases A, G, C, or T or analogs and they may be degenerated at one or more chosen nucleotide position(s). The primers or probes may be of any suitable length and may be selected anywhere within the DNA sequences from proprietary fragments or from selected database sequences which are suitable for (i) the universal detection of algae or archaea or bacteria or fingi or parasites, (ii) the species-specific detection and identification of any microorganism, including but not limited to: Abiotrophia adiacens, Bacteroides fragilis, Bordetella pertussis, Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Campylobacter jejuni and C. coli, Chlamydia pneumoniae, Chlamydia trachomatis, Cryptococcus neoformans, Cryptosporidium parvum, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Escherichia coli, Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Trypanosoma brucei, Trypanosoma cruzi, (iii) the genus-specific detection of Bordetella species, Candida species, Clostridium species, Corynebacterium species, Cryptococcus species, Entamoeba species, Enterococcus species, Gemella species, Giardia species, Legionella species, Leishmania species, Staphylococcus species, Streptococcus species, Trypanosoma species, (iv) the family-specific detection of Enterobacteriaceae family members, Mycobacteriaceae family members, Trypanosomatidae family members, (v) the detection of Enterococcus casseliflavus-flavescens-gallinarum group, Enterococcus, Gemella and Abiotrophia adiacens group, Pseudomonads extended group, Platelet-contaminating bacteria group, (vi) the detection of clinically important antimicrobial agents resistance genes listed in Table 5, (vii) the detection of clinically important toxin genes listed in Table 6.
Variants for a given target microbial gene are naturally occurring and are attributable to sequence variation within that gene during evolution (Watson et al., 1987, Molecular Biology of the Gene, 4th ed., The Benjamin/Cummings Publishing Company, Menlo Park, Calif., Lewin, 1989, Genes IV, John Wiley & Sons, New York, N.Y.). For example, different strains of the same microbial species may have a single or more nucleotide variation(s) at the oligonucleotide hybridization site. The person skilled in the art is well aware of the existence of variant algal, archaeal, bacterial, fungal or parasitical DNA nucleic acids and/or sequences for a specific gene and that the frequency of sequence variations depends on the selective pressure during evolution on a given gene product. The detection of a variant sequence for a region between two PCR primers may be demonstrated by sequencing the amplification product. In order to show the presence of sequence variants at the primer hybridization site, one has to amplify a larger DNA target with PCR primers outside that hybridization site. Sequencing of this larger fragment will allow the detection of sequence variation at this site. A similar strategy may be applied to show variants at the hybridization site of a probe. Insofar as the divergence of the target nucleic acids and/or sequences or a part thereof does not affect the specificity and ubiquity of the amplification primers or probes, variant microbial DNA is under the scope of this invention. Variants of the selected primers or probes may also be used to amplify or hybridize to a variant DNA.
Sequencing of tuf Nucleic Acids and/or Sequences from a Variety of Archaeal, Bacterial, Fungal and Parasitical Species
The nucleotide sequence of a portion of tuf nucleic acids and/or sequences was determined for a variety of archaeal, bacterial, fungal and parasitical species. The amplification primers (SEQ ID NOs. 664 and 697), which amplify a tuf gene portion of approximately 890 bp, were used along with newly designed sequencing primer pairs (See Annex I for the sequencing primers for tuf nucleic acids and/or sequences). Most primer pairs can amplify different copies of tuf genes (tufA and tufB). This is not surprising since it is known that for several bacterial species these two genes are nearly identical. For example, the entire tufA and tufB genes from E. coli differ at only 13 nucleotide positions (Neidhardt et al., 1996, Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed., American Society for Microbiology Press, Washington, D.C.). Similarly, some fungi are known to have two nearly identical copies of tuf nucleic acids and/or sequences (EF-1□). These amplification primers are degenerated at several nucleotide positions and contain inosines in order to allow the amplification of a wide range of tuf nucleic acids and/or sequences. The strategy used to select these amplification primers is similar to that illustrated in Annex I for the selection of universal primers. The tuf sequencing primers even sometimes amplified highly divergent copies of tuf genes (tufC) as illustrated in the case of some enterococcal species (SEQ ID NOs.: 73, 75, 76, 614 to 618, 621 and 987 to 989). To prove this, we have determined the enterococcal tuf nucleic acids and/or sequences from PCR amplicons cloned into a plasmid vector. Using the sequence data from the cloned amplicons, we designed new sequencing primers specific to the divergent (tufC) copy of enterococci (SEQ ID NOs.: 658-659 and 661) and then sequenced directly the tufC amplicons. The amplification primers (SEQ ID NOs.: 543, 556, 557, 643-645, 660, 664, 694, 696 and 697) could be used to amplify the tuf nucleic acids and/or sequences from any bacterial species. The amplification primers (SEQ ID NOs.: 558, 559, 560, 653, 654, 655, 813, 815, 1974-1984, 1999-2003) could be used to amplify the tuf (EF-1 genes from any fungal and/or parasitical species. The amplification primers SEQ ID NOs. 1221-1228 could be used to amplify bacterial tuf nucleic acids and/or sequences of the EF-G subdivision (fusA) (FIG. 3). The amplification primers SEQ ID NOs. 1224, and 1227-1229 could be used to amplify bacterial tuf nucleic acids and/or sequences comprising the end of EF-G (fusA) and the beginning of EF-Tu (tuf), including the intergenic region, as shown in FIG. 3.
Most tuf fragments to be sequenced were amplified using the following amplification protocol: One μl of cell suspension (or of purified genomic DNA 0.1-100 ng/μl) was transferred directly to 19 μl of a PCR reaction mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 1 μM of each of the 2 primers, 200 μM of each of the four dNTPs, 0.5 unit of Taq DNA polymerase (Promega Corp., Madison, Wis.). PCR reactions were subjected to cycling using a PTC-200 thermal cycler (MJ Research Inc., Watertown, Mass.) as follows: 3 min at 94-96° C. followed by 30-45 cycles of 1 min at 95° C. for the denaturation step, 1 min at 50-55° C. for the annealing step and 1 min at 72° C. for the extension step. Subsequently, twenty microliters of the PCR-amplified mixture were resolved by electrophoresis in a 1.5% agarose gel. The amplicons were then visualized by staining with methylene blue (Flores et al., 1992, Biotechniques, 13:203-205). The size of the amplification products was estimated by comparison with a 100-bp molecular weight ladder. The band corresponding to the specific amplification product was excised from the agarose gel and purified using the QIAquickTM gel extraction kit (QIAGEN Inc., Chatsworth, Calif.). The gel-purified DNA fragment was then used directly in the sequencing protocol. Both strands of the tuf genes amplification product were sequenced by the dideoxynucleotide chain termination sequencing method by using an Applied Biosystems automated DNA sequencer (model 377) with their Big DyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, Calif.). The sequencing reactions were performed by using the same amplification primers and 10 ng/100 bp of the gel-purified amplicon per reaction. For the sequencing of long amplicons such as those of eukaryotic tuf (EF-1 nucleic acids and/or sequences, we designed internal sequencing primers (SEQ ID NOs.: 654, 655 and 813) to be able to obtain sequence data on both strands for most of the fragment length. In order to ensure that the determined sequence did not contain errors attributable to the sequencing of PCR artefacts, we have sequenced two preparations of the gel-purified tuf amplification product originating from two independent PCR amplifications. For most target microbial species, the sequences determined for both amplicon preparations were identical. In case of discrepancies, amplicons from a third independent PCR amplification were sequenced. Furthermore, the sequences of both strands were 100% complementary thereby confirming the high accuracy of the determined sequence. The tuf nucleic acids and/or sequences determined using the above strategy are described in the Sequence Listing. Table 7 gives the originating microbial species and the source for each tuf sequence in the Sequence Listing.
The alignment of the tuf sequences determined by us or selected from databases revealed clearly that the length of the sequenced portion of the tuf genes is variable. There may be insertions or deletions of several amino acids. In addition, in several fungi introns were observed. Intron nucleic acids and/or sequences are part of tuf nucleic acids and/or sequences and could be useful in the design of species-specific primers and probes. This explains why the size of the sequenced tuf amplification products was variable from one fungal species to another. Consequently, the nucleotide positions indicated on top of each of Annexes IV to XX, XXIII to XXXI, XXXVIII and XLII do not correspond for sequences having insertions or deletions.
It should also be noted that the various tuf nucleic acids and/or sequences determined by us occasionally contain base ambiguities. These degenerated nucleotides correspond to sequence variations between tufA and tufB genes (or copies of the EF-G subdivision of tuf nucleic acids and/or sequences, or copies of EF-1- subdivision of tuf nucleic acids and/or sequences for fingi and parasites) because the amplification primers amplify both tuf genes. These nucleotide variations were not attributable to nucleotide misincorporations by the Taq DNA polymerase because the sequence of both strands was identical and also because the sequences determined with both preparations of the gel-purified tuf amplicons obtained from two independent PCR amplifications were identical.
The Selection of Amplification Primers from tuf Nucleic Acids and/or Sequences
The tuf sequences determined by us or selected from public databases were used to select PCR primers for universal detection of bacteria, as well as for genus-specific, species-specific family-specific or group-specific detection and identification. The strategy used to select these PCR primers was based on the analysis of multiple sequence alignments of various tuf sequences. For more details about the selection of PCR primers from tuf sequences please refer to Examples 5, 7-14, 17, 22, 24, 28, 30-31, 33, 36, and 38-40, and to Annexes VI-IX, XI-XIX and XXV.
Sequencing of atpD and recA Nucleic Acids and/or Sequences from a Variety of Archaeal, Bacterial, Fungal and Parasitical Species
The method used to obtain atpD and recA nucleic acids and/or sequences is similar to that described above for tuf nucleic acids and/or sequences.
The Selection of Amplification Primers from atpD or recA Nucleic Acids and/or Sequences
The comparison of the nucleotide sequence for the atpD or recA genes from various archaeal, bacterial, fungal and parasitical species allowed the selection of PCR primers (refer to Examples 6, 13, 29, 34 and 37, and to Annexes IV, V, X, and XX).
DNA Amplification
For DNA amplification by the widely used PCR (polymerase chain reaction) method, primer pairs were derived from proprietary DNA fragments or from database sequences. Prior to synthesis, the potential primer pairs were analyzed by using the OligoTM 5.0 software to verify that they were good candidates for PCR amplification.
During DNA amplification by PCR, two oligonucleotide primers binding respectively to each strand of the heat-denatured target DNA from the microbial genome are used to amplify exponentially in vitro the target DNA by successive thermal cycles allowing denaturation of the DNA, annealing of the primers and synthesis of new targets at each cycle (Persing et al, 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).
Briefly, the PCR protocols were as follows: Treated clinical specimens or standardized bacterial or fungal or parasitical suspensions (see below) or purified genomic DNA from bacteria, fungi or parasites were amplified in a 20 μl PCR reaction mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 2.5 mM MgCl2, 0.4 μM of each primer, 200 μM of each of the four dNTPs and 0.5 unit of Taq DNA polymerase (Promega) combined with the TaqStartTM antibody (Clontech Laboratories Inc., Palo Alto, Calif.). The TaqStartTM antibody, which is a neutralizing monoclonal antibody to Taq DNA polymerase, was added to all PCR reactions to enhance the specificity and the sensitivity of the amplifications (Kellogg et al., 1994, Biotechniques 16:1134-1137). The treatment of the clinical specimens varies with the type of specimen tested, since the composition and the sensitivity level required are different for each specimen type. It consists in a rapid protocol to lyse the microbial cells and eliminate or neutralize PCR inhibitors. For amplification from bacterial or fungal or parasitical cultures or from purified genomic DNA, the samples were added directly to the PCR amplification mixture without any pre-treatment step. An internal control was derived from sequences not found in the target microorganisms or in the human genome. The internal control was integrated into all amplification reactions to verify the efficiency of the PCR assays and to ensure that significant PCR inhibition was absent. Alternatively, an internal control derived from rRNA was also useful to monitor the efficiency of microbial lysis protocols.
PCR reactions were then subjected to thermal cycling (3 min at 94-96° C. followed by 30 cycles of 1 second at 95° C. for the denaturation step and 30 seconds at 50-65° C. for the annealing-extension step) using a PTC-200 thermal cycler (NJ Research Inc.). The number of cycles performed for the PCR assays varies according to the sensitivity level required. For example, the sensitivity level required for microbial detection directly from clinical specimens is higher for blood specimens than for urine specimens because the concentration of microorganisms associated with a septicemia can be much lower than that associated with a urinary tract infection. Consequently, more sensitive PCR assays having more thermal cycles are probably required for direct detection from blood specimens. Similarly, PCR assays performed directly from bacterial or fungal or parasitical cultures may be less sensitive than PCR assays performed directly from clinical specimens because the number of target organisms is normally much lower in clinical specimens than in microbial cultures.
The person skilled in the art of DNA amplification knows the existence of other rapid amplification procedures such as ligase chain reaction (LCR), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), branched DNA (bDNA), cycling probe technology (CPT), solid phase amplification (SPA), rolling circle amplification technology (RCA), solid phase RCA, anchored SDA and nuclease dependent signal amplification (NDSA) (Lee et al., 1997, Nucleic Acid Amplification Technologies: Application to Disease Diagnosis, Eaton Publishing, Boston, Mass.; Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.; Westin et al., 2000, Nat. Biotechnol. 18:199-204). The scope of this invention is not limited to the use of amplification by PCR, but rather includes the use of any rapid nucleic acid amplification method or any other procedure which may be used to increase the sensitivity and/or the rapidity of nucleic acid-based diagnostic tests. The scope of the present invention also covers the use of any nucleic acids amplification and detection technology including real-time or post-amplification detection technologies, any amplification technology combined with detection, any hybridization nucleic acid chips or arrays technologies, any amplification chips or combination of amplification and hybridization chips technologies. Detection and identification by any sequencing method is also under the scope of the present invention.
Any oligonucleotide suitable for the amplification of nucleic acids by approaches other than PCR or for DNA hybridization which are derived from the species-specific, genus-specific and universal DNA fragments as well as from selected antimicrobial agents resistance or toxin gene sequences included in this document are also under the scope of this invention.
Detection of Amplification Products
Classically, detection of amplification is performed by standard ethidium bromide-stained agarose gel electrophoresis. It is clear that other methods for the detection of specific amplification products, which may be faster and more practical for routine diagnosis, may be used. Such methods may be based on the detection of fluorescence after or during amplification. One simple method for monitoring amplified DNA is to measure its rate of formation by measuring the increase in fluorescence of intercalating agents such as ethidium bromide or SYBR® Green I (Molecular Probes). If more specific detection is required, fluorescence-based technologies can monitor the appearance of a specific product during the reaction. The use of dual-labeled fluorogenic probes such as in the TaqManTM system (Applied Biosystems) which utilizes the 5′-3′ exonuclease activity of the Taq polymerase is a good example (Livak K. J. et al. 1995, PCR Methods Appl. 4:357-362). TaqManTM can be performed during amplification and this “real-time” detection can be done in a single closed tube hence eliminating post-PCR sample handling and consequently preventing the risk of amplicon carryover. Several other fluorescence-based detection methods can be performed in real-time. Fluorescence resonance energy transfer (FRET) is the principle behind the use of adjacent hybridization probes (Wittwer, C. T. et al. 1997. BioTechniques 22:130-138), molecular beacons (Tyagi S, and Kramer F. R. 1996. Nature Biotechnology 14:303-308) and scorpions (Whitcomb et al. 1999. Nature Biotechnology 17:804-807). Adjacent hybridization probes are designed to be internal to the amplification primers. The 3′ end of one probe is labelled with a donor fluorophore while the 5′ end of an adjacent probe is labelled with an acceptor fluorophore. When the two probes are specifically hybridized in closed proximity (spaced by 1 to 5 nucleotides) the donor fluorophore which has been excited by an external light source emits light that is absorbed by a second acceptor that emit more fluorescence and yields a FRET signal. Molecular beacons possess a stem-and-loop structure where the loop is the probe and at the bottom of the stem a fluorescent moiety is at one end while a quenching moiety is at the other end. The beacons undergo a fluorogenic conformational change when they hybridize to their targets hence separating the fluorochrome from its quencher. The FRET principle is also used in an air thermal cycler with a built-in fluorometer (Wittwer, C. T. et al. 1997. BioTechniques 22:130-138). The amplification and detection are extremely rapid as reactions are performed in capillaries: it takes only 18 min to complete 45 cycles. Those techniques are suitable especially in the case where few pathogens are searched for. Boehringer-Roche Inc. sells the LightCyclerTM, and Cepheid makes the SmartCycler. These two apparatus are capable of rapid cycle PCR combined with fluorescent SYBR® Green I or FRET detection. We recently demonstrated in our laboratory, real-time detection of 10 CFU in less than 40 minutes using adjacent hybridization probes on the LightCyclerTM. Methods based on the detection of fluorescence are particularly promising for utilization in routine diagnosis as they are very rapid, quantitative and can be automated.
Microbial pathogens detection and identification may also be performed by solid support or liquid hybridization using species-specific internal DNA probes hybridizing to an amplification product. Such probes may be generated from any sequence from our repertory and designed to specifically hybridize to DNA amplification products which are objects of the present invention. Alternatively, the internal probes for species or genus or family or group detection and identification may be derived from the amplicons produced by a universal, family-, group-, genus- or species-specific amplification assay(s). The oligonucleotide probes may be labeled with biotin or with digoxigenin or with any other reporter molecule (for more details see below the section on hybrid capture). Hybrization on a solid support is amendable to miniaturization.
At present the oligonucleotide nucleic acid microarray technology is appealing. Currently, available low to medium density arrays (Heller et al., An integrated microelectronics hybridization system for genomic research and diagnostic applications. In: Harrison, D. J., and van den Berg, A., 1998, Micro total analysis systems '98, Kluwer Academic Publisher, Dordrecht.) could specifically capture fluorescent-labelled amplicons. Detection methods for hybridization are not limited to fluorescence; potentiometry, colorimetry and plasmon resonance are some examples of alternative detection methods. In addition to detection by hybridization, nucleic acid microarrays could be used to perform rapid sequencing by hybridization. Mass spectrometry could also be applicable for rapid identification of the amplicon or even for sequencing of the amplification products (Chiu and Cantor, 1999, Clinical Chemistry 45:1578; Berkenkamp et al., 1998, Science 281:260).
For the future of our assay format, we also consider the major challenge of molecular diagnostics tools, i.e.: integration of the major steps including sample preparation, genetic amplification, detection, data analysis and presentation (Anderson et al., Advances in integrated genetic analysis. In: Harrison, D. J., and van den Berg, A., 1998, Micro total analysis systems '98, Kluwer Academic Publisher, Dordrecht.).
To ensure PCR efficiency, glycerol, dimethyl sulfoxide (DMSO) or other related solvents can be used to increase the sensitivity of the PCR and to overcome problems associated with the amplification of a target DNA having a high GC content or forming strong secondary structures (Dieffenbach and Dveksler, 1995, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The concentration ranges for glycerol and DMSO are 5-15% (v/v) and 3-10% (v/v), respectively. For the PCR reaction mixture, the concentration ranges for the amplification primers and MgCl2 are 0.1-1.5 μM and 1.0-10.0 mM, respectively. Modifications of the standard PCR protocol using external and nested primers (i.e. nested PCR) or using more than one primer pair (i.e. multiplex PCR) may also be used (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). For more details about the PCR protocols and amplicon detection methods, see Examples.
Hybrid Capture and Chemiluminescence Detection of Amplification Products
Hybridization and detection of amplicons by chemiluminescence were adapted from Nikiforov et al. (1994, PCR Methods and Applications 3:285-291 and 1995, Anal. Biochem. 227:201-209) and from the DIGTM system protocol of Boehringer Mannheim. Briefly, 50 μl of a 25 picomoles solution of capture probe diluted in EDC {1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride} are immobilized in each well of 96-wells plates (MicroliteTM 2, Dynex) by incubation overnight at room temperature. The next day, the plates are incubated with a solution of 1% BSA diluted into TNTw (10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% TweenTM 20) for 1 hour at 37° C. The plates are then washed on a Wellwash AscentTM (Labsystems) with TNTw followed by Washing Buffer (100 mM maleic acid pH7.5; 150 mM NaCl; 0.3% TweenTM 20).
The amplicons were labelled with DIG-11-dUTP during PCR using the PCR DIG Labelling Mix from Boehringer Mannheim according to the manufacturer's instructions. Hybridization of the amplicons to the capture probes is performed in triplicate at stringent temperature (generally, probes are designed to allow hybrization at 55° C., the stringent temperature) for 30 minutes in 1.5 M NaCl; 10 mM EDTA. It is followed by two washes in 2×SSC; 0.1% SDS, then by four washes in 0.1×SSC; 0.1% SDS at the stringent temperature (55° C.). Detection with 1,2 dioxetane chemiluminescent alkaline phosphatase substrates like CSPD® (Tropix Inc.) is performed according to the manufacturer's instructions but with shorter incubations times and a different antibody concentration. The plates are agitated at each step, the blocking incubation is performed for only 5 minutes, the anti-DIG-AP1 is used at a 1:1000 dilution, the incubation with antibody lasts 15 minutes, the plates are washed twice for only 5 minutes. Finally, after a 2 minutes incubation into the detection buffer, the plates are incubated 5 minutes with CSPD® at room temperature followed by a 10 minutes incubation at 37° C. without agitation. Luminous signal detection is performed on a Dynex Microtiter Plate Luminometer using RLU (Relative Light Units).
Specificity, Ubiquity and Sensitivity Tests for Oligonucleotide Primers and Probes
The specificity of oligonucleotide primers and probes was tested by amplification of DNA or by hybridization with bacterial or fungal or parasitical species selected from a panel comprising closely related species and species sharing the same anatomo-pathological site (see Annexes and Examples). All of the bacterial, fungal and parasitical species tested were likely to be pathogens associated with infections or potential contaminants which can be isolated from clinical specimens. Each target DNA could be released from microbial cells using standard chemical and/or physical treatments to lyse the cells (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) or alternatively, genomic DNA purified with the GNOMETM DNA kit (Bio101, Vista, Calif.) was used. Subsequently, the DNA was subjected to amplification with the primer pairs. Specific primers or probes amplified only the target microbial species, genus, family or group.
Oligonucleotides primers found to amplify specifically the target species, genus, family or group were subsequently tested for their ubiquity by amplification (i.e. ubiquitous primers amplified efficiently most or all isolates of the target species or genus or family or group). Finally, the sensitivity of the primers or probes was determined by using 10-fold or 2-fold dilutions of purified genomic DNA from the targeted microorganism. For most assays, sensitivity levels in the range of 1-100 copies were obtained. The specificity, ubiquity and sensitivity of the PCR assays using the selected amplification primer pairs were tested either directly from cultures of microbial species or from purified microbial genomic DNA.
Probes were tested in hybrid capture assays as described above. An oligonucleotide probe was considered specific only when it hybridized solely to DNA from the species or genus or family or group from which it was selected. Oligonucleotide probes found to be specific were subsequently tested for their ubiquity (i.e. ubiquitous probes detected efficiently most or all isolates of the target species or genus or family or group) by hybridization to microbial DNAs from different clinical isolates of the species or genus or family or group of interest including ATCC reference strains. Similarly, oligonucleotide primers and probes could be derived from antimicrobial agents resistance or toxin genes which are objects of the present invention.
Reference Strains
The reference strains used to build proprietary tuf, atpD and recA sequence data subrepertories, as well as to test the amplification and hybridization assays were obtained from (i) the American Type Culture Collection (ATCC), (ii) the Laboratoire de santé publique du Québec (LSPQ), (iii) the Centers for Disease Control and Prevention (CDC), (iv) the National Culture Type Collection (NCTC) and (v) several other reference laboratories throughout the world. The identity of our reference strains was confirmed by phenotypic testing and reconfirmed by analysis of tuf, atpD and recA sequences (see Example 13).
Antimicrobial Agents Resistance Genes
Antimicrobial resistance complicates treatment and often leads to therapeutic failures. Furthermore, overuse of antibiotics inevitably leads to the emergence of microbial resistance. Our goal is to provide clinicians, in approximately one hour, the needed information to prescribe optimal treatments. Besides the rapid identification of negative clinical specimens with DNA-based tests for universal algal, archaeal, bacterial, fungal or parasitical detection and the identification of the presence of a specific pathogen in the positive specimens with species- and/or genus- and/or family- and/or group-specific DNA-based tests, clinicians also need timely information about the ability of the microbial pathogen to resist antibiotic treatments. We feel that the most efficient strategy to evaluate rapidly microbial resistance to antimicrobials is to detect directly from the clinical specimens the most common and clinically important antimicrobial agents resistance genes (i.e. DNA-based tests for the specific detection of antimicrobial agents resistance genes). Since the sequence from the most important and common antimicrobial agents resistance genes are available from public databases, our strategy is to use the sequence from a portion or from the entire resistance gene to design specific oligonucleotide primers or probes which will be used as a basis for the development of sensitive and rapid DNA-based tests. The list of each of the antimicrobial agents resistance genes selected on the basis of their clinical relevance (i.e. high incidence and importance) is given in Table 5; descriptions of the designed amplification primers and internal probes are given in Annexes XXXIV-XXXVII, XXXIX, XLV, and L-LI. Our approach is unique because the antimicrobial agents resistance genes detection and the microbial detection and identification can be performed simultaneously, or independently, or sequentially in multiplex or parallel or sequential assays under uniform PCR amplification conditions. These amplifications can also be done separately.
Toxin Genes
Toxin identification is often very important to prescribe optimal treatments. Besides the rapid identification of negative clinical specimens with DNA-based tests for universal bacterial detection and the identification of the presence of a specific pathogen in the positive specimens with species- and/or genus- and/or family- and/or group-specific DNA-based tests, clinicians sometimes need timely information about the ability of certain bacterial pathogens to produce toxins. Since the sequence from the most important and common bacterial toxin genes are available from public databases, our strategy is to use the sequence from a portion or from the entire toxin gene to design specific oligonucleotide primers or probes which will be used as a basis for the development of sensitive and rapid DNA-based tests. The list of each of the bacterial toxin genes selected on the basis of their clinical relevance (i.e. high incidence and importance) is given in Table 6; descriptions of the designed amplification primers and internal probes are given in Annexes XXII, XXXII and XXXIII. Our approach is unique because the toxin genes detection and the bacterial detection and identification can be performed simultaneously, or independently, or sequentially, in multiplex or parallel or sequential assays under uniform PCR amplification conditions. These amplifications can also be done separately.
Universal Bacterial Detection
In the routine microbiology laboratory, a high percentage of clinical specimens sent for bacterial identification are negative by culture. Testing clinical samples with universal amplification primers or universal probes to detect the presence of bacteria prior to specific identification and screening out the numerous negative specimens is thus useful as it reduces costs and may rapidly orient the clinical management of the patients. Several amplification primers and probes were therefore synthesized from highly conserved portions of bacterial sequences from the tuf, atpD and recA nucleic acids and/or sequences. The universal primers selection was based on a multiple sequence alignment constructed with sequences from our repertory.
All computer analysis of amino acid and nucleotide sequences were performed by using the GCG programs. Subsequently, optimal PCR primers for the universal amplification of bacteria were selected with the help of the OligoTM program. The selected primers are degenerated at several nucleotide positions and contain several inosines in order to allow the amplification of all clinically relevant bacterial species. Inosine is a nucleotide analog able to specifically bind to any of the four nucleotides A, C, G or T. Degenerated oligonucleotides consist of an oligonucleotide mix having two or more of the four nucleotides A, C, G or T at the site of mismatches. The inclusion of inosine and/or of base ambiguities in the amplification primers allow mismatch tolerance thereby permitting the amplification of a wider array of target nucleotide sequences (Dieffenbach and Dveksler, 1995 PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
The amplification conditions with the universal primers are very similar to those used for the species- and genus-specific amplification assays except that the annealing temperature is slightly lower. The original universal PCR assay described in our assigned WO98/20157 (SEQ ID NOs. 23-24 of the latter application) was specific and nearly ubiquitous for the detection of bacteria. The specificity for bacteria was verified by amplifying genomic DNA isolated from the 12 fungal species as well as genomic DNA from Leishmania donovani, Saccharomyces cerevisiae and human lymphocytes. None of the above eukaryotic DNA preparations could be amplified by the universal assay, thereby suggesting that this test is specific for bacteria. The ubiquity of the universal assay was verified by amplifying genomic DNAs from 116 reference strains which represent 95 of the most clinically relevant bacterial species. These species have been selected from the bacterial species listed in Table 4. We found that at least 104 of these strains could be amplified. However, the assay could be improved since bacterial species which could not be amplified with the original tuf nucleic acids and/or sequences-based assay included species belonging to the following genera: Corynebacterium (11 species) and Stenotrophomonas (1 species). Sequencing of the tuf genes from these bacterial species and others has been performed in the scope of the present invention in order to improve the universal assay. This sequencing data has been used to select new universal primers which may be more ubiquitous and more sensitive. Also, we improved our primer and probes design strategy by taking into consideration the phylogeny observed in analysing our repertory of tuf, atpD and recA sequences. Data from each of the 3 main subrepertories (tuf atpD and recA) was subjected to a basic phylogenic analysis using the Pileup command from version 10 of the GCG package (Genetics Computer Group, inc.). This analysis indicated the main branches or phyla reflecting the relationships between sequences. Instead of trying to design primers or probes able to hybridize to all phyla, we designed primers or probes able to hybridize to the main phyla while trying to use the largest phylum possible. This strategy should allow less degenerated primers hence improving sensitivity and by combining primers in a multiplex assay, improve ubiquity. Universal primers SEQ ID NOs. 643-645 based on tuf sequences have been designed to amplify most pathogenic bacteria except Actinomyceteae, Clostridiaceae and the Cytophaga, Flexibacter and Bacteroides phylum (pathogenic bacteria of this phylum include mostly Bacteroides, Porphyromonas and Prevotella species). Primers to fill these gaps have been designed for Actinomyceteae (SEQ ID NOs. 646-648), Clostridiaceae (SEQ ID NOs. 796-797, 808-811), and the Cytophaga, Flexibacter and Bacteroides phylum (SEQ ID NOs. 649-651), also derived from tuf nucleic acids and/or sequences. These primers sets could be used alone or in conjuction to render the universal assay more ubiquitous.
Universal primers derived from atpD sequences include SEQ ID NOs. 562-565. Combination of these primers does not amplify human DNA but should amplify almost all pathogenic bacterial species except proteobacteria belonging to the epsilon subdivision (Campylobacter and Helicobacter), the bacteria from the Cytophaga, Flexibacter and Bacteroides group and some actinomycetes and corynebacteria. By analysing atpD sequences from the latter species, primers and probes to specifically fill these gaps could be designed and used in conjuction with primers SEQ ID NOs. 562-565, also derived from atpD nucleic acids and/or sequences.
In addition, universality of the assay could be expanded by mixing atpD sequences-derived primers with tuf sequences-derived primers. Ultimately, even recA sequences-derived primers could be added to fill some gaps in the universal assay.
It is important to note that the 95 bacterial species selected to test the ubiquity of the universal assay include all of the most clinically relevant bacterial species associated with a variety of human infections acquired in the community or in hospitals (nosocomial infections). The most clinically important bacterial and fungal pathogens are listed in Tables 1 and 2.
Amino Acid Sequences Derived from tuf, atpD and recA Nucleic Acids and/or Sequences
The amino acid sequences translated from the repertory of tuf, atpD and recA nucleic acids and/or sequences are also an object of the present invention. The amino acid sequence data will be particularly useful for homology modeling of three-dimensional (3D) structure of the elongation factor Tu, elongation factor G, elongation factor 1α, ATPase subunit beta and RecA recombinase. For all these proteins, at least one structure model has been published using X-ray diffraction data from crystals. Based on those structural informations it is possible to use computer software to build 3D model structures for any other protein having peptide sequence homologies with the known structure (Greer, 1991, Methods in Enzymology, 202:239-252; Taylor, 1994, Trends Biotechnol., 12(5):154-158; Sali, 1995, Curr. Opin. Biotechnol. 6:437-451; Sanchez and Sali, 1997, Curr. Opin. Struct. Biol. 7:206-214; Fischer and Eisenberg, 1999, Curr. Opin. Struct. Biol. 9:208-211; Guex et al., 1999, Trends Biochem. Sci. 24: 364-367). Model structures of target proteins are used for the design or to predict the behavior of ligands and inhibitors such as antibiotics. Since EF-Tu and EF-G are already known as antibiotic targets (see above) and since the beta subunit of ATPase and RecA recombinase are essential to the survival of the microbial cells in natural conditions of infection, all four proteins could be considered antibiotic targets. Sequence data, especially the new data generated by us could be very useful to assist the creation of new antibiotic molecules with desired spectrum of activity. In addition, model structures could be used to improve protein function for commercial purposes such as improving antibiotic production by microbial strains or increasing biomass.
The following detailed embodiments and appended drawings are provided as illustrative examples of his invention, with no intention to limit the scope thereof.
Examples and Annexes
For sake of clarity, here is a list of Examples and Annexes:
Example 1: Sequencing of bacterial atpD (F-type and V-type) gene fragments.
Example 2: Sequencing of eukaryotic atpD (F-type and V-type) gene fragments.
Example 3: Sequencing of eukaryotic tuf (EF-1) gene fragments.
Example 4: Sequencing of eukaryotic tuf (organelle origin, M) gene fragments.
Example 5: Specific detection and identification of Streptococcus agalactiae using tuf sequences.
Example 6: Specific detection and identification of Streptococcus agalactiae using atpD sequences.
Example 7: Development of a PCR assay for detection and identification of staphylococci at genus and species levels.
Example 8: Differentiating between the two closely related yeast species Candida albicans and Candida dubliniensis. 
Example 9: Specific detection and identification of Entamoeba histolytica. 
Example 10: Sensitive detection and identification of Chlamydia trachomatis. 
Example 11: Genus-specific detection and identification of enterococci.
Example 12: Detection and identification of the major bacterial platelets contaminants using tuf sequences with a multiplex PCR test.
Example 13: The resolving power of the tuf and atpD sequences databases is comparable to the biochemical methods for bacterial identification.
Example 14: Detection of group B streptococci from clinical specimens.
Example 15: Simultaneous detection and identification of Streptococcus pyogenes and its pyrogenic exotoxin A.
Example 16: Real-time detection and identification of Shiga toxin-producing bacteria.
Example 17: Development of a PCR assay for the detection and identification of staphylococci at genus and species levels and its associated mecA gene.
Example 18: Sequencing of pbp1a, pbp2b and pbp2x genes of Streptoccoccus pneumoniae. 
Example 19: Sequencing of hexA genes of Streptococcus species.
Example 20: Development of a multiplex PCR assay for the detection of Streptococcus pneumoniae and its penicillin resistance genes.
Example 21: Sequencing of the vancomycin resistance vanA, vanC1, vanC2 and vanC3 genes.
Example 22: Development of a PCR assay for the detection and identification of enterococci at genus and species levels and its associated resistance genes vanA and vanB.
Example 23: Development of a multiplex PCR assay for detection and identification of vancomycin-resistant Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens. 
Example 24: Universal amplification involving the EF-G (fusA) subdivision of tuf sequences.
Example 25: DNA fragment isolation from Staphylococcus saprophyticus by arbitrarily primed PCR.
Example 26: Sequencing of prokaryotic tuf gene fragments.
Example 27: Sequencing of procaryotic recA gene fragments.
Example 28: Specific detection and identification of Escherichia coli/Shigella sp. using tuf sequences.
Example 29: Specific detection and identification of Klebsiella pneumoniae using atpD sequences.
Example 30: Specific detection and identification of Acinetobacter baumanii using tuf sequences.
Example 31: Specific detection and identification of Neisseria gonorrhoeae using tuf sequences.
Example 32: Sequencing of bacterial gyrA and parC gene fragments.
Example 33: Development of a PCR assay for the specific detection and identification of Staphylococcus aureus and its quinolone resistance genes gyrA and parC.
Example 34: Development of a PCR assay for the detection and identification of Klebsiella pneumoniae and its quinolone resistance genes gyrA and parC.
Example 35: Development of a PCR assay for the detection and identification of Streptococcus pneumoniae and its quinolone resistance genes gyrA and parC.
Example 36: Detection of extended-spectrum TEM-type β-lactamases in Escherichia coli. 
Example 37: Detection of extended-spectrum SHV-type β-lactamases in Klebsiella pneumoniae. 
Example 38: Development of a PCR assay for the detection and identification of Neisseria gonorrhoeae and its associated tetracycline resistance gene tetM.
Example 39: Development of a PCR assay for the detection and identification of Shigella sp. and their associated trimethoprim resistance gene dhfr1a.
Example 40: Development of a PCR assay for the detection and identification of Acinetobacter baumanii and its associated aminoglycoside resistance gene aph(3′)-VIa.
Example 41: Specific detection and identification of Bacteroides fragilis using atpD (V-type) sequences.
Example 42: Evidence for horizontal gene transfer in the evolution of the elongation factor Tu in Enterococci.
Example 43: Elongation factor Tu (tuf) and the F-ATPase beta-subunit (atpD) as phylogenetic tools for species of the family Enterobacteriaceae.
Example 44: Testing new pairs of PCR primers selected from two species-specific genomic DNA fragments which are objects of U.S. Pat. No. 6,001,564.
Example 45: Testing modified versions of PCR primers derived from the sequence of several primers which are objects of U.S. Pat. No. 6,001,564.
The various Annexes show the strategies used for the selection of a variety of DNA amplification primers, nucleic acid hybridization probes and molecular beacon internal probes:

    
    
        (i) Annex I shows the amplification primers used for nucleic acid amplification from tuf sequences.
        (ii) Annex II shows the amplification primers used for nucleic acid amplification from atpD sequences.
        (iii) Annex III shows the internal hybridization probes for detection of tuf sequences.
        (iv) Annex IV illustrates the strategy used for the selection of the amplification primers specific for atpD sequences of the F-type.
        (v) Annex V illustrates the strategy used for the selection of the amplification primers specific for atpD sequences of the V-type.
        (vi) Annex VI illustrates the strategy used for the selection of the amplification primers specific for the tuf sequences of organelle lineage (M, the letter M is used to indicate that in most cases, the organelle is the mitochondria).
        (vii) Annex VII illustrates the strategy used for the selection of the amplification primers specific for the tuf sequences of eukaryotes (EF-1).
        (viii) Annex VII illustrates the strategy for the selection of Streptococcus agalactiae-specific amplification primers from tuf sequences.
        (ix) Annex IX illustrates the strategy for the selection of Streptococcus agalactiae-specific hybridization probes from tuf sequences.
        (x) Annex X illustrates the strategy for the selection of Streptococcus agalactiae-specific amplification primers from atpD sequences.
        (xi) Annex XI illustrates the strategy for the selection from tuf sequences of Candida albicans/dubliniensis-specific amplification primers, Candida albicans-specific hybridization probe and Candida dubliniensis-specific hybridization probe.
        (xii) Annex XII illustrates the strategy for the selection of Staphylococcus-specific amplification primers from tuf sequences.
        (xiii) Annex XIII illustrates the strategy for the selection of the Staphylococcus-specific hybridization probe from tuf sequences.
        (xiv) Annex XIV illustrates the strategy for the selection of Staphylococcus saprophyticus-specific and Staphylococcus haemolyticus-specific hybridization probes from tuf sequences.
        (xv) Annex XV illustrates the strategy for the selection of Staphylococcus aureus-specific and Staphylococcus epidermidis-specific hybridization probes from tuf sequences.
        (xvi) Annex XVI illustrates the strategy for the selection of the Staphylococcus hominis-specific hybridization probe from tuf sequences.
        (xvii) Annex XVII illustrates the strategy for the selection of the Enterococcus-specific amplification primers from tuf sequences.
        (xviii) Annex XVIII illustrates the strategy for the selection of the Enterococcus faecalis-specific hybridization probe, of the Enterococcus faecium-specific hybridization probe and of the Enterococcus casseliflavus-flavescens-gallinarum group-specific hybridization probe from tuf sequences.
        (xix) Annex XIX illustrates the strategy for the selection of primers from tuf sequences for the identification of platelets contaminants.
        (xx) Annex XX illustrates the strategy for the selection of the universal amplification primers from atpD sequences.
        (xxi) Annex XXI shows the amplification primers used for nucleic acid amplification from recA sequences.
        (xxii) Annex XXII shows the specific and ubiquitous primers for nucleic acid amplification from speA sequences.
        (xxiii) Annex XXIII illustrates the first strategy for the selection of Streptococcus pyogenes-specific amplification primers from speA sequences.
        (xxiv) Annex XXIV illustrates the second strategy for the selection of Streptococcus pyogenes-specific amplification primers from speA sequences.
        (xxv) Annex XXV illustrates the strategy for the selection of Streptococcus pyogenes-specific amplification primers from tuf sequences.
        (xxvi) Annex XXVI illustrates the strategy for the selection of stx1-specific amplification primers and hybridization probe.
        (xxvii) Annex XXVII illustrates the strategy for the selection of stx2-specific amplification primers and hybridization probe.
        (xxviii) Annex XXVIII illustrates the strategy for the selection of vanA-specific amplification primers from van sequences.
        (xxix) Annex XXIX illustrates the strategy for the selection of vanB-specific amplification primers from van sequences.
        (xxx) Annex XXX illustrates the strategy for the selection of vanC-specific amplification primers from vanC sequences.
        (xxxi) Annex XXXI illustrates the strategy for the selection of Streptococcus pneumoniae-specific amplification primers and hybridization probes from pbp1a sequences.
        (xxxii) Annex XXXII shows the specific and ubiquitous primers for nucleic acid amplification from toxin gene sequences.
        (xxxiii) Annex XXXIII shows the molecular beacon internal hybridization probes for specific detection of toxin sequences.
        (xxxiv) Annex XXXIV shows the specific and ubiquitous primers for nucleic acid amplification from van sequences.
        (xxxv) Annex XXXV shows the internal hybridization probes for specific detection of van sequences.
        (xxxvi) Annex XXXVI shows the specific and ubiquitous primers for nucleic acid amplification from pbp sequences.
        (xxxvii) Annex XXXVII shows the internal hybridization probes for specific detection of pbp sequences.
        (xxxviii) Annex XXXVIII illustrates the strategy for the selection of vanAB-specific amplification primers and vanA- and vanB-specific hybridization probes from van sequences.
        (xxxix) Annex XXXIX shows the internal hybridization probe for specific detection of mecA.
        (xl) Annex XL shows the specific and ubiquitous primers for nucleic acid amplification from hexA sequences.
        (xli) Annex XLI shows the internal hybridization probe for specific detection of hexA.
        (xlii) Annex XLII illustrates the strategy for the selection of Streptococcus pneumoniae species-specific amplification primers and hybridization probe from hexA sequences.
        (xliii) Annex XLIII shows the specific and ubiquitous primers for nucleic acid amplification from pcp sequences.
        (xliv) Annex XLIV shows specific and ubiquitous primers for nucleic acid amplification of S. saprophyticus sequences of unknown coding potential.
        (xlv) Annex XLV shows the molecular beacon internal hybridization probes for specific detection of antimicrobial agents resistance gene sequences.
        (xlvi) Annex XLVI shows the molecular beacon internal hybridization probe for specific detection of S. aureus gene sequences of unknown coding potential.
        (xlvii) Annex XLVII shows the molecular beacon hybridization internal probe for specific detection of tuf sequences.
        (xlviii) Annex XLVIII shows the molecular beacon internal hybridization probes for specific detection of ddl and mtl sequences.
        (xlix) Annex XLIX shows the internal hybridization probe for specific detection of S. aureus sequences of unknown coding potential.
        (l) Annex L shows the amplification primers used for nucleic acid amplification from antimicrobial agents resistance genes sequences.
        (li) Annex LI shows the internal hybridization probes for specific detection of antimicrobial agents resistance genes sequences.
        (lii) Annex LII shows the molecular beacon internal hybridization probes for specific detection of atpD sequences.
        (liii) Annex LIII shows the internal hybridization probes for specific detection of atpD sequences.
        (liv) Annex LIVI shows the internal hybridization probes for specific detection of ddI and mtl sequences.
    
    


As shown in these Annexes, the selected amplification primers may contain inosines and/or base ambiguities. Inosine is a nucleotide analog able to specifically bind to any of the four nucleotides A, C, G or T. Alternatively, degenerated oligonucleotides which consist of an oligonucleotide mix having two or more of the four nucleotides A, C, G or T at the site of mismatches were used. The inclusion of inosine and/or of degeneracies in the amplification primers allows mismatch tolerance thereby permitting the amplification of a wider array of target nucleotide sequences (Dieffenbach and Dveksler, 1995 PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
EXAMPLES
Example 1
Sequencing of Bacterial atpD (F-Type and V-Type) Gene Fragments.
As shown in Annex IV, the comparison of publicly available atpD (F-type) sequences from a variety of bacterial species revealed conserved regions allowing the design of PCR primers able to amplify atpD sequences (F-type) from a wide range of bacterial species. Using primers pairs SEQ ID NOs. 566 and 567, 566 and 814, 568 and 567, 570 and 567, 572 and 567, 569 and 567, 571 and 567, 700 and 567, it was possible to amplify and sequence atpD sequences SEQ ID NOs. 242-270, 272-398, 673-674, 737-767, 866-867, 942-955, 1245-1254, 1256-1265, 1527, 1576, 1577, 1600-1604, 1640-1646, 1649, 1652, 1655, 1657, 1659-1660, 1671, 1844-1845, and 1849-1865.
Similarly, Annex V shows the strategy to design the PCR primers able to amplify atpD sequences of the V-type from a wide range of archaeal and bacterial species. Using primers SEQ ID NOs. 681-683, it was possible to amplify and sequence atpD sequences SEQ ID NOs. 827-832, 929-931, 958 and 966. As the gene was difficult to amplify for several species, additional amplification primers were designed inside the original amplicon (SEQ ID NOs. 1203-1207) in order to obtain sequence information for these species. Other primers (SEQ ID NO. 1212, 1213, 2282-2285) were also designed to amplify regions of the atpD gene (V-type) in archaebacteria.
Example 2
Sequencing of Eukaryotic atpD (F-Type and V-Type) Gene Fragments.
The comparison of publicly available atpD (F-type) sequences from a variety of fungal and parasitical species revealed conserved regions allowing the design of PCR primers able to amplify atpD sequences from a wide range of fungal and parasitical species. Using primers pairs SEQ ID NOs. 568 and 573, 574 and 573, 574 and 708, and 566 and 567, it was possible to amplify and sequence atpD sequences SEQ ID NOs. 458-497, 530-538, 663, 667, 676, 678-680, 768-778, 856-862, 889-896, 941, 1638-1639, 1647, 1650-1651, 1653-1654, 1656, 1658, 1684, 1846-1848, and 2189-2192.
In the same manner, the primers described in Annex V (SEQ ID NOs. 681-683) could amplify the atpD (V-type) gene from various fungal and parasitical species. This strategy allowed to obtain SEQ ID NOs. 834-839, 956-957, and 959-965.
Example 3
Sequencing of Eukaryotic tuf (EF-1) Gene Fragments.
As shown in Annex VII, the comparison of publicly available tuf (EF-1) sequences from a variety of fungal and parasitical species revealed conserved regions allowing the design of PCR primers able to amplify tuf sequences from a wide range of fungal and parasitical species. Using primers pairs SEQ ID NOs. 558 and 559, 813 and 559, 558 and 815, 560 and 559, 653 and 559, 558 and 655, and 654 and 559, 1999 and 2000, 2001 and 2003, 2002 and 2003, it was possible to amplify and sequence tuf sequences SEQ ID NOs. 399-457, 509-529, 622-624, 677, 779-790, 840-842, 865, 897-903, 1266-1287, 1561-1571 and 1685.
Example 4
Sequencing of Eukaryotic tuf (Organelle Origin, M) Gene Fragments.
As shown in Annex VI, the comparison of publicly available tuf (organelle origin, M) sequences from a variety of fungal and parasitical organelles revealed conserved regions allowing the design of PCR primers able to amplify tuf sequences of several organelles belonging to a wide range fungal and parasitical species. Using primers pairs SEQ ID NOs. 664 and 652, 664 and 561, 911 and 914, 912 and 914, 913 and 915, 916 and 561, 664 and 917, it was possible to amplify and sequence tuf sequences SEQ ID NOs. 498-508, 791-792, 843-855, 904-910, 1664, 1666-1667, 1669-1670, 1673-1683, 1686-1689, 1874-1876, 1879, 1956-1960, and 2193-2199.
Example 5
Specific Detection and Identification of Streptococcus agalactiae Using tuf Sequences.
As shown in Annex VIII, the comparison of tuf sequences from a variety of bacterial species allowed the selection of PCR primers specific for S. agalactiae. The strategy used to design the PCR primers was based on the analysis of a multiple sequence alignment of various tuf sequences. The multiple sequence alignment includes the tuf sequences of four bacterial strains from the target species as well as tuf sequences from other species and bacterial genera, especially representatives of closely related species. A careful analysis of this alignment allowed the selection of oligonucleotide sequences which are conserved within the target species but which discriminate sequences from other species and genera, especially from the closely related species, thereby permitting the species-specific, ubiquitous and sensitive detection and identification of the target bacterial species.
The chosen primer pair, oligos SEQ ID NO. 549 and SEQ ID NO. 550, gives an amplification product of 252 bp. Standard PCR was carried out using 0.4 μM of each primer, 2.5 mM MgCl2, BSA 0.05 mM, 1×Taq Buffer (Promega), dNTP 0.2 mM (Pharmacia), 0.5 U Taq DNA polymerase (Promega) coupled with TaqStartTM antibody (Clontech Laboratories Inc., Palo Alto), 1 μl of genomic DNA sample in a final volume of 20 μl using a PTC-200 thermocycler (MJ Research Inc.). The optimal cycling conditions for maximum sensitivity and specificity were 3 minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 62° C., followed by terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide.
Specificity of the assay was tested by adding into the PCR reactions, 0.1 ng of genomic DNA from each of the bacterial species listed in Table 8. Efficient amplification was observed only for the 5 S. agalactiae strains listed. Of the other bacterial species, including 32 species representative of the vaginal flora and 27 other streptococcal species, only S. acidominimus yielded amplification. The signal with 0.1 ng of S. acidominimus genomic DNA was weak and the detection limit for this species was 10 pg (corresponding to more than 4000 genome copies) while the detection limit for S. agalactiae was 2.5 fg (corresponding to one genome copy) of genomic DNA.
To increase the specificity of the assay, internal probes were designed for FRET (Fluorescence Resonance Energy Transfer) detection using the LightCyclerTM (Idaho Technology). As illustrated in Annex IX, a multiple sequence alignment of streptococcal tuf sequence fragments corresponding to the 252 bp region amplified by primers SEQ ID NO. 549 and SEQ ID NO. 550, was used for the design of internal probes TSagHF436 (SEQ ID NO. 582) and TSagHF465 (SEQ ID NO. 583). The region of the amplicon selected for internal probes contained sequences unique and specific to S. agalactiae. SEQ ID NO. 583, the more specific probe, is labelled with fluorescein in 3′, while SEQ ID NO. 582, the less discriminant probe, is labelled with CY5 in 5′ and blocked in 3′ with a phosphate group. However, since the FRET signal is only emitted if both probes are adjacently hybridized on the same target amplicon, detection is highly specific.
Real-time detection of PCR products using the LightCyclerTM was carried out using 0.4 μM of each primer (SEQ ID NO. 549-550), 0.2 μM of each probe (SEQ ID NO. 582-583), 2.5 mM MgCl2, BSA 450 μg/ml, 1×PC2 Buffer (AB Peptides, St-Louis, Mo.), dNTP 0.2 mM (Pharmacia), 0.5 U KlenTaq1TM DNA polymerase (AB Peptides) coupled with TaqStartTM antibody (Clontech Laboratories Inc., Palo Alto), 0.7 μl of genomic DNA sample in a final volume of 7 μl using a LightCycler thermocycler (Idaho Technology). The optimal cycling conditions for maximum sensitivity and specificity were 3 minutes at 94° C. for initial denaturation, then forty cycles of three steps consisting of 0 second (this setting meaning the LightCycler will reach the target temperature and stay at it for its minimal amount of time) at 94° C., 10 seconds at 64° C., 20 seconds at 72° C. Amplification was monitored during each annealing steps using the fluorescence ratio. The streptococcal species having close sequence homologies with the tuf sequence of S. agalactiae (S. acidominimus, S. anginosus, S. bovis, S. dysgalactiae, S. equi, S. ferns, S. gordonii, S. intermedius, S. parasanguis, S. parauberis, S. salivarius, S. sanguis, S. suis) as well as S. agalactiae were tested in the LightCycler with 0.07 ng of genomic DNA per reaction. Only S. agalactiae yielded an amplification signal, hence demonstrating that the assay is species-specific. With the LightCyclerTM assay using the internal FRET probes, the detection limit for S. agalactiae was 1-2 genome copies of genomic DNA.
Example 6
Specific Detection and Identification of Streptococcus agalactiae Using atpD Sequences.
As shown in Annex X, the comparison of atpD sequences from a variety of bacterial species allowed the selection of PCR primers specific for S. agalactiae. The primer design strategy is similar to the strategy described in the preceding Example except that atpD sequences were used in the alignment.
Four primers were selected, ASag42 (SEQ ID NO. 627), ASag52 (SEQ ID NO. 628), ASag206 (SEQ ID NO. 625) and ASag371 (SEQ ID NO. 626). The following combinations of these four primers give four amplicons; SEQ ID NO. 627+SEQ ID NO. 625=190 bp, SEQ ID NO. 628+SEQ ID NO. 625=180 bp, SEQ ID NO. 627+SEQ ID NO. 626=355 bp, and SEQ ID NO. 628+SEQ ID NO. 626=345 bp.
Standard PCR was carried out on PTC-200 thermocyclers (MJ Research Inc) using 0.4 μM of each primers pair, 2.5 mM MgCl2, BSA 0.05 mM, 1×taq Buffer (Promega), dNTP 0.2 mM (Pharmacia), 0.5 U Taq DNA polymerase (Promega) coupled with TaqStartTM antibody (Clontech Laboratories Inc., Palo Alto), 1 μl of genomic DNA sample in a final volume of 20 μL. The optimal cycling conditions for maximum sensitivity and specificity were adjusted for each primer pair. Three minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at the optimal annealing temperature specified below were followed by terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. Since atpD sequences are relatively more specific than tuf sequences, only the most closely related species namely, the steptococcal species listed in Table 9, were tested.
All four primer pairs only amplified the six S. agalactiae strains. With an annealing temperature of 63° C., the primer pair SEQ ID NO. 627+SEQ ID NO. 625 had a sensitivity of 1-5 fg (equivalent to 1-2 genome copies). At 55° C., the primer pair SEQ ID NO. 628+SEQ ID NO. 625 had a sensitivity of 2.5 fg (equivalent to 1 genome copy). At 60° C., the primer pair SEQ ID NO. 627+SEQ ID NO. 626 had a sensitivity of 10 fg (equivalent to 4 genome copies). At 58° C., the primer pair SEQ ID NO. 628+SEQ ID NO. 626 had a sensitivity of 2.5-5 fg (equivalent to 1-2 genome copies). This proves that all four primer pairs can detect S. agalactiae with high specificity and sensitivity. Together with Example 5, this example demonstrates that both tuf and atpD sequences are suitable and flexible targets for the identification of microorganisms at the species level. The fact that 4 different primer pairs based on atpD sequences led to efficient and specific amplification of S. agalactiae demonstrates that the challenge is to find target genes suitable for diagnostic purposes, rather than finding primer pairs from these target sequences.
Example 7
Development of a PCR Assay for Detection and Identification of Staphylococci at Genus. and Species Levels
Materials and Methods
Bacterial strains. The specificity of the PCR assay was verified by using a panel of ATCC (America Type Culture Collection) and DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; German Collection of Microorganisms and Cell Cultures) reference strains consisting of 33 gram-negative and 47 gram-positive bacterial species (Table 12). In addition, 295 clinical isolates representing 11 different species of staphylococci from the microbiology laboratory of the Centre Hospitalier Universitaire de Québec , Pavillon Centre Hospitalier de l'Université Laval (CHUL) (Step-Foy, Québec , Canada) were also tested to further validate the Staphylococcus-specific PCR assay. These strains were all identified by using (i) conventional methods or (ii) the automated MicroScan Autoscan-4 system equipped with the Positive BP Combo Panel Type 6 (Dade Diagnostics, Mississauga, Ontario, Canada). Bacterial strains from frozen stocks kept at −80° C. in brain heart infusion (BHI) broth containing 10% glycerol were cultured on sheep blood agar or in BHI broth (Quelab Laboratories Inc, Montreal, Québec , Canada).
PCR primers and internal probes. Based on multiple sequence alignments, regions of the tuf gene unique to staphylococci were identified. Staphylococcus-specific PCR primers TStaG422 (SEQ ID NO. 553) and TStaG765 (SEQ ID NO. 575) were derived from these regions (Annex XII). These PCR primers are displaced by two nucleotide positions compared to original Staphylococcus-specific PCR primers described in our patent publication WO98/20157 (SEQ ID NOs. 17 and 20 in the said patent publication). These modifications were done to ensure specificity and ubiquity of the primer pair, in the light of new tuf sequence data revealed in the present patent application for several additional staphylococcal species and strains.
Similarly, sequence alignment analysis were performed to design genus and species-specific internal probes (see Annexes XIII to XVI). Two internal probes specific for Staphylococcus (SEQ ID NOs. 605-606), five specific for S. aureus (SEQ ID NOs. 584-588), five specific for S. epidermidis (SEQ ID NO. 589-593), two specific for S. haemolyticus (SEQ ID NOs. 594-595), three specific for S. hominis (SEQ ID NOs. 596-598), four specific for S. saprophyticus (SEQ ID NOs. 599-601 and 695), and two specific for coagulase-negative Staphylococcus species including S. epidermidis, S. hominis, S. saprophyticus, S. auricularis, S. capitis, S. haemolyticus, S. lugdunensis, S. simulans, S. cohnii and S. warneri (SEQ ID NOs. 1175-1176) were designed. The range of mismatches between the Staphylococcus-specific 371-bp amplicon and each of the 20-mer species-specific internal probes was from 1 to 5, in the middle of the probe when possible. No mismatches were present in the two Staphylococcus-specific probes for the 11 species analyzed: S. aureus, S. auricularis, S. capitis, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, S. saprophyticus, S. simulans and S. warneri. In order to verify the intra-specific sequence conservation of the nucleotide sequence, sequences were obtained for the 371-bp amplicon from five unrelated ATCC and clinical strains for each of the species S. aureus, S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus. The OligoTM (version 5.0) primer analysis software (National Biosciences, Plymouth, Minn.) was used to confirm the absence of self-complementary regions within and between the primers or probes. When required, the primers contained inosines or degenerated nucleotides at one or more variable positions. Oligonucleotide primers and probes were synthesized on a model 394 DNA synthesizer (Applied Biosystems, Mississauga, Ontario, Canada). Detection of the hybridization was performed with the DIG-labeled dUTP incorporated during amplification with the Staphylococcus-specific PCR assay, and the hybridization signal was detected with a luminometer (Dynex Technologies) as described above in the section on luminescent detection of amplification products. Annexes XIII to XVI illustrate the strategy for the selection of several internal probes.
PCR amplification. For all bacterial species, amplification was performed from purified genomic DNA or from a bacterial suspension whose turbidity was adjusted to that of a 0.5 McFarland standard, which corresponds to approximately 1.5×108 bacteria per ml. One nanogram of genomic DNA or 1 □l of the standardized bacterial suspension was transferred directly to a 19 □l PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.2 □M (each) of the two Staphylococcus genus-specific primers (SEQ ID NOs. 553 and 575), 200 □M (each) of the four deoxynucleoside triphosphates (Pharmacia Biotech), 3.3 □g/□l bovine serum albumin (BSA) (Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada), and 0.5 U Taq polymerase (Promega) coupled with TaqStartTM Antibody (Clontech). The PCR amplification was performed as follows: 3 min. at 94° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 55° C., plus a terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. Visualization of the PCR products was made under UV at 254 nm.
For determination of the sensitivities of the PCR assays, two-fold dilutions of purified genomic DNA were used to determine the minimal number of genome copies which can be detected.
Results
Amplifications with the Staphylococcus genus-specific PCR assay. The specificity of the assay was assessed by performing 30-cycle and 40-cycle PCR amplifications with the panel of gram-positive (47 species from 8 genera) and gram-negative (33 species from 22 genera) bacterial species listed in Table 12. The PCR assay was able to detect efficiently 27 of 27 staphylococcal species tested in both 30-cycle and 40-cycle regimens. For 30-cycle PCR, all bacterial species tested other than staphylococci were negative. For 40-cycle PCR, Enterococcus faecalis and Macrococcus caseolyticus were slightly positive for the Staphylococcus-specific PCR assay. The other species tested remained negative. Ubiquity tests performed on a collection of 295 clinical isolates provided by the microbiology laboratory of the Centre Hospitalier Universitaire de Québec , Pavillon Centre Hospitalier de l'Université Laval (CHUL), including Staphylococcus aureus (n=34), S. auricularis (n=2), S. capitis (n=19), S. cohnii (n=5), S. epidermidis (n=18), S. haemolyticus (n=21), S. hominis (n=73), S. lugdunensis (n=17), S. saprophyticus (n=6), S. simulans (n=3), S. warneri (n=32) and Staphylococcus sp. (n=65), showed a uniform amplification signal with the 30-cycle PCR assays and a perfect relation between the genotype and classical identification schemes.
The sensitivity of the Staphylococcus-specific assay with 30-cycle and 40-cycle PCR protocols was determined by using purified genomic DNA from the 11 staphylococcal species previously mentioned. For PCR with 30 cycles, a detection limit of 50 copies of genomic DNA was consistently obtained. In order to enhance the sensitivity of the assay, the number of cycles was increased. For 40-cycle PCR assays, the detection limit was lowered to a range of 5-10 genome copies, depending on the staphylococcal species tested.
Hybridization between the Staphylococcus-specific 371-bp amplicon and species-specific or genus-specific internal probes. Inter-species polymorphism was sufficient to generate species-specific internal probes for each of the principal species involved in human diseases (S. aureus, S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus). In order to verify the intra-species sequence conservation of the nucleotide sequence, sequence comparisons were performed on the 371-bp amplicon from five unrelated ATCC and clinical strains for each of the 5 principal staphylococcal species: S. aureus, S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus. Results showed a high level of conservation of nucleotide sequence between different unrelated strains from the same species. This sequence information allowed the development of staphylococcal species identification assays using species-specific internal probes hybridizing to the 371-bp amplicon. These assays are specific and ubiquitous for those five staphylococcal species. In addition to the species-specific internal probes, the genus-specific internals probes were able to recognize all or most Staphylococcus species tested.
Example 8
Differentiating Between the Two Closely Related Yeast Species Candida albicans and Candida dubliniensis. 
It is often useful for the clinician to be able to differentiate between two very closely related species of microorganisms. Candida albicans is the most important cause of invasive human mycose. In recent years, a very closely related species, Candida dubliniensis, was isolated in immunosuppressed patients. These two species are difficult to distinguish by classic biochemical methods. This example demonstrates the use of tuf sequences to differentiate Candida albicans and Candida dubliniensis. PCR primers SEQ ID NOs. 11-12, from previous patent publication WO98/20157, were selected for their ability to specifically amplify a tuf (elongation factor 1 alpha type) fragment from both species (see Annex XI for primer positions). Within this tuf fragment, a region differentiating C. albicans and C. dubliniensis by two nucleotides was selected and used to design two internal probes (see Annex XI for probe design, SEQ ID NOs. 577 and 578) specific for each species. Amplification of genomic DNA from C. albicans and C. dubliniensis was carried out using DIG-11-dUTP as described above in the section on chemiluminescent detection of amplification products. Internal probes SEQ ID NOs. 577 and 578 were immobilized on the bottom of individual microtiter plates and hybridization was carried out as described above in the above section on chemiluminescent detection of amplification products. Luminometer data showed that the amplicon from C. albicans hybridized only to probe SEQ ID NO. 577 while the amplicon from C. dubliniensis hybridized only to probe SEQ ID NO. 578, thereby demonstrating that each probe was species-specific.
Example 9
Specific Identification of Entamoeba histolytica. 
Upon analysis of tuf (elongation factor 1 alpha) sequence data, it was possible to find four regions where Entamoeba histolytica sequences remained conserved while other parasitical and eukaryotic species have diverged. Primers TEntG38 (SEQ ID NO. 703), TEntG442 (SEQ ID NO. 704), TEntG534 (SEQ ID NO. 705), and TEntG768 (SEQ ID NO. 706) were designed so that SEQ ID NO. 703 could be paired with the three other primers. On PTC-200 thermocyclers (MJ Research), the cycling conditions for initial sensitivity and specificity testing were 3 min. at 94° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 55° C., followed by terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. The three primer pairs could detect the equivalent of less than 200 E. histolytica genome copies. Specificity was tested using 0.5 ng of purified genomic DNA from a panel of microorganisms including Babesia bovis, Babesia microtti, Candida albicans, Crithidia fasciculata, Leishmania major, Leishmania hertigi and Neospora caninum. Only E. histolytica DNA could be amplified, thereby suggesting that the assay was species-specific.
Example 10
Sensitive Identification of Chlamydia trachomatis. 
Upon analysis of tuf sequence data, it was possible to find two regions where Chlamydia trachomatis sequences remained conserved while other species have diverged. Primers Ctr82 (SEQ ID NO. 554) and Ctr249 (SEQ ID NO. 555) were designed. With the PTC-200 thermocyclers (MJ Research), the optimal cycling conditions for maximum sensitivity and specificity were determined to be 3 min. at 94° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 60° C., followed by terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. The assay could detect the equivalent of 8 C. trachomatis genome copies. Specificity was tested with 0.1 ng of purified genomic DNA from a panel of microorganisms including 22 species commonly encountered in the vaginal flora (Bacillus subtilis, Bacteroides fragilis, Candida albicans, Clostridium difficile, Corynebacterium cervicis, Corynebacterium urealyticum, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus influenzae, Klebsiella oxytoca, Lactobacillus acidophilus, Peptococcus niger, Peptostreptococcus prevotii, Porphyromonas asaccharolytica, Prevotella melaminogenica, Propionibacterium acnes, Staphylococcus aureus, Streptococcus acidominimus, and Streptococcus agalactiae). Only C. trachomatis DNA could be amplified, thereby suggesting that the assay was species-specific.
Example 11
Genus-Specific Detection and Identification of Enterococci.
Upon analysis of tuf sequence data and comparison with the repertory of tuf sequences, it was possible to find two regions where Enterococcus sequences remained conserved while other genera have diverged (Annex XVII). Primer pair Encg313dF and Encg599c (SEQ ID NOs. 1137 and 1136) was tested for its specificity by using purified genomic DNA from a panel of bacteria listed in Table 10. Using the PTC-200 thermocycler (MJ Research), the optimal cycling conditions for maximum sensitivity and specificity were determined to be 3 min. at 94° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 55° C., followed by terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. Visualization of the PCR products was made under UV at 254 nm. The 18 enterococcal species listed in Table 10 were all amplified efficiently. The only other species amplified were Abiotrophia adiacens, Gemella haemolysans and Gemella morbillorum, three gram-positive species. Sensitivity tested with several strains of E. casseliflavus, E. faecium, E. faecalis, E. flavescens and E. gallinarum and with one strain of each other Enterococcus species listed in Table 10 ranged from 1 to 10 copies of genomic DNA. The sequence variation within the 308-bp amplicon was sufficient so that internal probes could be used to speciate the amplicon and differenciate enterococci from Abiotrophia adiacens, Gemella haemolysans and Gemella morbillorum, thereby allowing to achieve excellent specificity. Species-specific internal probes were generated for each of the clinically important species, E. faecalis (SEQ ID NO. 1174), E. faecium (SEQ ID NO. 602), and the group including E. casseliflavus, E. flavescens and E. gallinarum (SEQ ID NO. 1122) (Annex XVIII). The species-specific internal probes were able to differentiate their respective Enterococcus species from all other Enterococcus species. These assays are sensitive, specific and ubiquitous for those five Enterococcus species.
Example 12
Identification of the Major Bacterial Platelets Contaminants Using tuf Sequences with a Multiplex PCR Test.
Blood platelets preparations need to be monitored for bacterial contaminations. The tuf sequences of 17 important bacterial contaminants of platelets were aligned. As shown in Annex XIX, analysis of these sequences allowed the design of PCR primers. Since in the case of contamination of platelet concentrates, detecting all species (not just the more frequently encountered ones) is desirable, perfect specificity of primers was not an issue in the design. However, sensitivity is important. That is why, to avoid having to put too much degeneracy, only the most frequent contaminants were included in primer design, knowing that the selected primers would anyway be able to amplify more species than the 17 used in the design because they target highly conserved regions of tuf sequences. Oligonucleotide sequences which are conserved in these 17 major bacterial contaminants of platelet concentrates were chosen (oligos Tplaq 769 and Tplaq 991, respectively SEQ ID NOs. 636 and 637) thereby permitting the detection of these bacterial species. However, sensitivity was slightly deficient with staphylococci. To ensure maximal sensitivity in the detection of all the more frequent bacterial contaminants, a multiplex assay also including oligonucleotide primers targetting the Staphylococcus genera (oligos Stag 422, SEQ ID NO. 553; and Stag 765, SEQ ID NO. 575) was developed. The bacterial species detected with the assay are listed in Table 14.
The primer pairs, oligos SEQ ID NO. 636 and SEQ ID NO. 637 that give an amplification product of 245 pb, and oligos SEQ ID NO. 553 and SEQ ID NO. 575 that give an amplification product of 368 pb, were used simultaneously in the multiplex PCR assay. Detection of these PCR products was made on the LightCycler thermocycler (Idaho Technology) using SYBR® Green I (Molecular Probe Inc.). SYBR® Green I is a fluorescent dye that binds specifically to double-stranded DNA.
Fluorogenic detection of PCR products with the LightCycler was carried out using 1.0 μM of both Tplaq primers (SEQ ID NOs. 636-637) and 0.4 μM of both TStaG primers (SEQ ID NOs. 553 and 575), 2.5 mM MgCl2, BSA 7.5 μM, dNTP 0.2 mM (Pharmacia), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.5 U Taq DNA polymerase (Boerhinger Mannheim) coupled with TaqStartTM antibody (Clontech), and 0.07 ng of genomic DNA sample in a final volume of 7 μl. The optimal cycling conditions for maximum sensitivity and specificity were 1 minute at 94° C. for initial denaturation, then forty-five cycles of three steps consisting of 0 second at 95° C., 5 seconds at 60° C. and 9 seconds at 72° C. Amplification was monitored during each elongation cycle by measuring the level of SYBR® Green I. However, real analysis takes place after PCR. Melting curves are done for each sample and transformation of the melting peak allows determination of Tm. Thus primer-dimer and specific PCR product are discriminated. With this assay, all prominent bacterial contaminants of platelet concentrates listed in Annex XIX and Table 14 were detected. Sensitivity tests were performed on the 9 most frequent bacterial contaminants of platelets. The detection limit was less than 20 genome copies for E. cloacae, B. cereus, S. choleraesuis and S. marcescens; less than 15 genome copies for P. aeruginosa; and 2 to 3 copies were detected for S. aureus, S. epidermidis, E. coli and K. pneumoniae. Further refinements of assay conditions should increase sensitivity levels.
Example 13
The Resolving Power of the tuf and atpD Sequences DataBases is Comparable to the Biochemical Methods for Bacterial Identification.
The present gold standard for bacterial identification is mainly based on key morphological traits and batteries of biochemical tests. Here we demonstrate that the use of tuf and atpD sequences combined with simple phylogenetic analysis of databases formed by these sequences is comparable to the gold standard. In the process of acquiring data for the tuf sequences, we sequenced the tuf gene of a strain that was given to us labelled as Staphylococcus hominis ATCC 35982. That tuf sequence (SEQ ID NO. 192) was incorporated into the tuf sequences database and subjected to a basic phylogenic analysis using the Pileup command from version 10 of the GCG package (Genetics Computer Group). This analysis indicated that SEQ ID NO. 192 is not associated with other S. hominis strains but rather with the S. warneri strains. The ATCC 35982 strain was sent to the reference laboratory of the Laboratoire de santé publique du Québec (LSPQ). They used the classic identification scheme for staphylococci (Kloos and Schleifer, 1975, J. Clin. Microbiol. 1:82-88). Their results shown that although the colonial morphology could correspond to S. hominis, the more precise biochemical assays did not. These assays included discriminant mannitol, mannose and ribose acidification tests as well as rapid and dense growth in deep thioglycolate agar. The LSPQ report identified strain ATCC 35982 as S. warneri which confirms our database analysis. The same thing happened for S. warneri (SEQ ID NO. 187) which had initially been identified as S. haemolyticus by a routine clinical laboratory using a low resolving power automated system (MicroScan, AutoScan-4TM). Again, the tuf and LSPQ analysis agreed on its identification as S. warneri. In numerous other instances, in the course of acquiring tuf and atpD sequence data from various species and genera, analysis of our tuf and/or atpD sequence databases permitted the exact identification of mislabelled or erroneously identified strains. These results clearly demonstrate the usefulness and the high resolving power of our sequence-based identification assays using the tuf and atpD sequences databases.
Example 14
Detection of Group B Streptococci from Clinical Specimens.*
Introduction
Streptococcus agalactiae, the group B streptococcus (GBS), is responsible for a severe illness affecting neonate infants. The bacterium is passed from the healthy carrier mother to the baby during delivery. To prevent this infection, it is recommended to treat expectant mothers susceptible of carrying GBS in their vaginal/anal flora. Carrier status is often a transient condition and rigorous monitoring requires cultures and classic bacterial identification weeks before delivery. To improve the detection and identification of GBS we developed a rapid, specific and sensitive PCR test fast enough to be performed right at delivery.
Materials and Methods
GBS clinical specimens. A total of 66 duplicate vaginal/anal swabs were collected from 41 consenting pregnant women admitted for delivery at the Centre Hospitalier Universitaire de Québec , Pavillon Saint-François d'Assise following the CDC recommendations. The samples were obtained either before or after rupture of membranes. The swab samples were tested at the Centre de Recherche en Infectiologie de l'Université Laval within 24 hours of collection. Upon receipt, one swab was cut and then the tip of the swab was added to GNS selective broth for identification of group B streptococci (GBS) by the standard culture methods recommended by the CDC. The other swab was processed following the instruction of the IDI DNA extraction kit (Infectio Diagnostics (IDI) Inc.) prior to PCR amplification.
Oligonucleotides. PCR primers, Tsag340 (SEQ ID NO. 549) and Tsag552 (SEQ ID NO. 550) complementary to the regions of the tuf gene unique for GBS were designed based upon a multiple sequence alignment using our repertory of tuf sequences. Oligo primer analysis software (version 5.0) (National Biosciences) was used to analyse primers annealing temperature, secondary structure potential as well as mispriming and dimerization potential. The primers were synthesized using a model 391 DNA synthesizer (Applied Biosystems).
A pair of fluorescently labeled adjacent hybridization probes Sag465-F (SEQ ID NO. 583) and Sag436-C (SEQ ID NO. 582) were synthesized and purified by Operon Technologies. They were designed to meet the recommendations of the manufacturer (Idaho Technology) and based upon multiple sequence alignment analysis using our repertory of tuf sequences to be specific and ubiquitous for GBS. These adjacent probes, which are separated by one nucleotide, allow fluorescence resonance energy transfer (FRET), generating an increased fluorescence signal when both hybridized simultaneously to their target sequences. The probe SEQ ID NO. 583 was labeled with FITC in 3 prime while SEQ ID NO. 582 was labeled with Cy5 in 5 prime. The Cy5-labeled probes contained a 3′-blocking phosphate group to prevent extension of the probes during the PCR reactions.
PCR amplification. Conventional amplifications were performed either from 2 μl of a purified genomic DNA preparation or cell lysates of vaginal/anal specimens. The 20 μl PCR mixture contained 0.4 μM of each GBS-specific primer (SEQ ID NOs. 549-550), 200 μM of each deoxyribonucleotide (Pharmacia Biotech), 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 3.3 mg/ml bovine serum albumin (BSA) (Sigma), and 0.5 U of Taq polymerase (Promega) combined with the TaqStartTM antibody (Clontech). The TaqStartTM antibody, which is a neutralizing monoclonal antibody of Taq DNA polymerase, was added to all PCR reactions to enhance the efficiency of the amplification. The PCR mixtures were subjected to thermal cycling (3 min at 95° C. and then 40 cycles of 1 s at 95° C., and 30 s at 62° C. with a 2-min final extension at 72° C.) with a PTC-200 DNA Engine thermocycler (MJ research). The PCR-amplified reaction mixture was resolved by agarose gel electrophoresis.
The LightCyclerTM PCR amplifications were performed with 1 μl of a purified genomic DNA preparation or cell lysates of vaginal/anal specimens. The 10 μl amplification mixture consisted of 0.4 μM each GBS-specific primer (SEQ ID NOs. 549-550), 200 μM each dNTP, 0.2 μM each fluorescently labeled probe (SEQ ID NOs. 582-583), 300 μg/ml BSA (Sigma), and 1 μl of 10×PC2 buffer (containing 50 mM Tris-HCl (pH 9.1), 16 mM ammonium sulfate, 3.5 mM Mg2+, and 150 μg/ml BSA) and 0.5 U KlenTaq1TM (AB Peptides) coupled with TaqStartTM antibody (Clontech). KlenTaq1TM is a highly active and more heat-stable DNA polymerase without 5′-exonuclease activity. This prevents hydrolysis of hybridized probes by the 5′ to 3′ exonuclease activity. A volume of 7 μl of the PCR mixture was transferred into a composite capillary tube (Idaho Technology). The tubes were then centrifuged to move the reaction mixture to the tips of the capillaries and then cleaned with optical-grade methanol. Subsequently the capillaries were loaded into the carousel of a LC32 LightCyclerTM (Idaho Technology), an instrument that combines rapid-cycle PCR with fluorescence analysis for continuous monitoring during amplification. The PCR reaction mixtures were subjected to a denaturation step at 94° C. for 3 min followed by 45 cycles of 0 s at 94° C., 20 s at 64° C. and 10 s at 72° C. with a temperature transition rate of 20° C./s. Fluorescence signals were obtained at each cycle by sequentially positioning each capillary on the carousel at the focus of optical elements affiliated to the built-in fluorimeter for 100 milliseconds. Complete amplification and analysis required about 35 min.
Specificity and sensitivity tests. The specificity of the conventional and LightCyclerTM PCR assays was verified by using purified genomic DNA (0.1 ng/reaction) from a battery of ATCC reference strains representing 35 clinically relevant gram-positive species (Abiotrophia defectiva ATCC 49176, Bifidobacterium breve ATCC 15700, Clostridium difficile ATCC 9689, Corynebacterium urealyticum ATCC 43042Enterococcus casseliflavus ATCC 25788, Enterococcus durans ATCC 19432, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 19434, Enterococcus gallinarum ATCC 49573, Enterococcus raffinosus ATCC 49427, Lactobacillus reuteri ATCC 23273, Lactococcus lactis ATCC 19435, Listeria monocytogenes ATCC 15313, Peptococcus niger ATCC 27731, Peptostreptococcus anaerobius ATCC 27337, Peptostreptococcus prevotii ATCC 9321, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 14990, Staphylococcus haemolyticus ATCC 29970, Staphylococcus saprophyticus ATCC 15305, Streptococcus agalactiae ATCC 27591, Streptococcus anginosus ATCC 33397, Streptococcus bovis ATCC 33317, Streptococcus constellatus ATCC 27823, Streptococcus dysgalactiae ATCC 43078, Streptococcus gordonii ATCC 10558, Streptococcus mitis ATCC 33399, Streptococcus mutans ATCC 25175, Streptococcus oralis ATCC 35037, Streptococcus parauberis ATCC 6631, Streptococcus pneumoniae ATCC 6303, Streptococcus pyogenes ATCC 19615, Streptococcus salivarius ATCC 7073, Streptococcus sanguinis ATCC 10556, Streptococcus uberis ATCC 19436). These microbial species included 15 species of streptococci and many members of the normal vaginal and anal floras. In addition, 40 GBS isolates of human origin, whose identification was confirmed by a latex agglutination test (Streptex, Murex), were also used to evaluate the ubiquity of the assay.
For determination of the sensitivities (i.e., the minimal number of genome copies that could be detected) for conventional and LightCyclerTM PCR assays, serial 10-fold or 2-fold dilutions of purified genomic DNA from 5 GBS ATCC strains were used.
Results
Evaluation of the GBS-specific conventional and LightCyclerTM PCR assays. The specificity of the two assays demonstrated that only DNAs from GBS strains could be amplified. Both PCR assays did not amplify DNAs from any other bacterial species tested including 14 streptococcal species other than GBS as well as phylogenetically related species belonging to the genera Enterococcus, Peptostreptococcus and Lactococcus. Important members of the vaginal or anal flora, including coagulase-negative staphylococci, Lactobacillus sp., and Bacteriodes sp. were also negative with the GBS-specific PCR assay. The LightCyclerTM PCR assays detected only GBS DNA by producing an increased fluorescence signal which was interpreted as a positive PCR result. Both PCR methods were able to amplify all of 40 GBS clinical isolates, showing a perfect correlation with the phenotypic identification methods.
The sensitivity of the assay was determined by using purified genomic DNA from the 5 ATCC strains of GBS. The detection limit for all of these 5 strains was one genome copy of GBS. The detection limit of the assay with the LightCyclerTM was 3.5 fg of genomic DNA (corresponding to 1-2 genome copies of GBS). These results confirmed the high sensitivity of our GBS-specific PCR assay.
Direct Detection of GBS from vaginal/anal specimens. Among 66 vaginal/anal specimens tested, 11 were positive for GBS by both culture and PCR. There was one sample positive by culture only. The sensitivity of both PCR methods with vaginal/anal specimens for identifying colonization status in pregnant women at delivery was 91.7% when compared to culture results. The specificity and positive predictive values were both 100% and the negative predictive value was 97.8%. The time for obtaining results was approximately 45 min for LightCyclerTM PCR, approximately 100 min for conventional PCR and 48 hours for culture.
Conclusion
We have developed two PCR assays (conventional and LightCyclerTM) for the detection of GBS, which are specific (i.e., no amplification of DNA from a variety of bacterial species other than GBS) and sensitive (i.e., able to detect around 1 genome copy for several reference ATCC strains of GBS). Both PCR assays are able to detect GBS directly from vaginal/anal specimens in a very short turnaround time. Using the real-time PCR assay on LightCyclerTM, we can detect GBS carriage in pregnant women at delivery within 45 minutes.
Example 15
Simultaneous Detection and Identification of Streptococcus pyogenes and its Pyrogenic Exotoxin A.
The rapid detection of Streptococcus pyogenes and of its pyrogenic exotoxin A is of clinical importance. We developed a multiplex assay which permits the detection of strains of S. pyogenes carrying the pyrogenic toxin A gene, which is associated with scarlet fever and other pathologies. In order to specifically detect S. pyogenes, nucleotide sequences of the pyrrolidone carboxylyl peptidase (pcp) gene were aligned to design PCR primers Spy291 (SEQ ID NO. 1211) and Spy473 (SEQ ID NO. 1210). Next, we designed primers for the specific detection of the pyrogenic exotoxin A. Nucleotide sequences of the speA gene, carried on the bacteriophage T12, were aligned as shown in Annex XXIII to design PCR primers Spytx814 (SEQ ID NO. 994) and Spytx 927 (SEQ ID NO. 995).
The primer pairs: oligos SEQ ID NOs. 1210-1211, yielding an amplification product of 207 bp, and oligos SEQ ID NOs. 994-995, yielding an amplification product of 135 bp, were used in a multiplex PCR assay.
PCR amplification was carried out using 0.4 μM of both pairs of primers, 2.5 mM MgCl2, BSA 0.05 μM, dNTP 0.2 μM (Pharmacia), 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.5 U Taq DNA polymerase (Promega) coupled with TaqStartTM antibody (Clontech Laboratories Inc.), and 1 μl of genomic DNA sample in a final volume of 20 μl. PCR amplification was performed using a PTC-200 thermal cycler (MJ Research). The optimal cycling conditions for maximum specificity and sensitivity were 3 minutes at 94° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 63° C., followed by a final step of 2 minutes at 72° C. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. Visualization of the PCR products was made under UV at 254 nm.
The detection limit was less than 5 genome copies for both S. pyogenes and its pyrogenic exotoxin A. The assay was specific for pyrogenic exotoxin A-producing S. pyogenes: strains of the 27 other species of Streptococcus tested, as well as 20 strains of various gram-positive and gram-negative bacterial species were all negative.
A similar approach was used to design an alternative set of speA-specific primers (SEQ ID NOs. 996 to 998, see Annex XXIV). In addition, another set of primers based on the tuf gene (SEQ ID NOs. 999 to 1001, see Annex XXV) could be used to specifically detect Streptococcus pyogenes. 
Example 16
Real-Time Detection and Identification of Shiga Toxin-Producing Bacteria.
Shiga toxin-producing Escherichia coli and Shigella dysenteriae cause bloody diarrhea. Currently, identification relies mainly on the phenotypic identification of S. dysenteriae and E. coli serotype O157:H7. However, other serotypes of E. coli are increasingly found to be producers of type 1 and/or type 2 Shiga toxins. Two pairs of PCR primers targeting highly conserved regions present in each of the Shiga toxin genes stx1 and stx2 were designed to amplify all variants of those genes (see Annexes XXVI and XXVII). The first primer pair, oligonucleotides 1SLT224 (SEQ ID NO. 1081) and 1SLT385 (SEQ ID NO. 1080), yields an amplification product of 186 bp from the stx1 gene. For this amplicon, the 1SLTB1-Fam (SEQ ID NO. 1084) molecular beacon was designed for the specific detection of stx1 using the fluorescent label 6-carboxy-fluorescein. The 1SltS1-FAM (SEQ ID NO. 2012) molecular scorpion was also designed as an alternate way for the specific detection of stx1. A second pair of PCR primers, oligonucleotides 2SLT537 (SEQ ID NO. 1078) and 2SLT678b (SEQ ID NO. 1079), yields an amplification product of 160 bp from the stx2 gene. Molecular beacon 2SLTB1-Tet (SEQ ID NO. 1085) was designed for the specific detection of stx2 using the fluorescent label 5-tetrachloro-fluorescein. Both primer pairs were combined in a multiplex PCR assay.
PCR amplification was carried out using 0.8 μM of primer pair SEQ ID NOs. 1080-1081, 0.5 μM of primer pair SEQ ID NOs. 1078-1079, 0.3 μM of each molecular beacon, 8 mM MgCl2, 490 μg/mL BSA, 0.2 mM dNTPs (Pharmacia), 50 mM Tris-HCl, 16 mM NH4SO4, 1× TaqMaster (Eppendorf), 2.5 U KlenTaq1 DNA polymerase (AB Peptides) coupled with TaqStartTM antibody (Clontech Laboratories Inc.), and 1 μl of genomic DNA sample in a final volume of 25 μl. PCR amplification was performed using a SmartCycler thermal cycler (Cepheid). The optimal cycling conditions for maximum sensitivity and specificity were 60 seconds at 95° C. for initial denaturation, then 45 cycles of three steps consisting of 10 seconds at 95° C., 15 seconds at 56° C. and 5 seconds at 72° C. Detection of the PCR products was made in real-time by measuring the fluorescent signal emitted by the molecular beacon when it hybridizes to its target at the end of the annealing step at 56° C.
The detection limit was the equivalent of less than 5 genome copies. The assay was specific for the detection of both toxins, as demonstrated by the perfect correlation between PCR results and the phenotypic characterization performed using antibodies specific for each Shiga toxin type. The assay was successfully performed on several Shiga toxin-producing strains isolated from various geographic areas of the world, including 10 O157:H7 E. coli, 5 non-O157:H7 E. coli and 4 S. dysenteriae. 
Example 17
Development of a PCR Assay for the Detection and Identification of Staphylococci at Genus and Species Levels and its Associated mecA Gene.
The Staphylococcus-specific PCR primers described in Example 7 (SEQ ID NOs. 553 and 575) were used in multiplex with the mecA-specific PCR primers and the S. aureus-specific primers described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and 262 for mecA and SEQ ID NOs. 152 and 153 for S. aureus in the said patent). Sequence alignment analysis of 10 publicly available mecA gene sequences allowed to design an internal probe specific to mecA (SEQ ID NO. 1177). An internal probe was also designed for the S. aureus-specific amplicon (SEQ ID NO 1234). PCR amplification and agarose gel electrophoresis of the amplified products were performed as described in Example 7, with the exception that 0.4 μM (each) of the two Staphylococcus-specific primers (SEQ ID NOs. 553 and 575) and 0.4 μM (each) of the mecA-specific primers and 0.4 μM (each) of the S. aureus-specific primers were used in the PCR mixture. The specificity of the multiplex assay with 40-cycle PCR protocols was verified by using purified genomic DNA from five methicillin-resistant and fifteen methicillin-sensitive staphylococcal strains. The sensitivity of the multiplex assay with 40-cycle PCR protocols was determined by using purified genomic DNA from twenty-three methicillin-resistant and twenty-eight methicillin-sensitive staphylococcal strains. The detection limit was 2 to 10 genome copies of genomic DNA, depending on the staphylococcal species tested. Furthermore, the mecA-specific internal probe, the S. aureus-specific internal probe and the coagulase-negative staphylococci-specific internal probe (described in Example 7) were able to recognize twenty-three methicillin-resistant staphylococcal strains and twenty-eight methicillin-sensitive staphylococcal strains with high sensitivity and specificity.
The format of the assay is not limited to the one described above. A person skilled in the art could adapt the assay for different formats such as PCR with real-time detection using molecular beacon probes. Molecular beacon probes designed to be used in this assay include, but are not limited to, SEQ ID NO. 1232 for detection of the S. aureus-specific amplicon, SEQ ID NO. 1233 for detection of coagulase-negative staphylococci and SEQ ID NO. 1231 for detection of mecA.
Alternatively, a multiplex PCR assay containing the Staphylococcus-specific PCR primers described in Example 7 (SEQ ID NOs. 553 and 575) and the mecA-specific PCR primers described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and 262 in the said patent) were developed. PCR amplification and agarose gel electrophoresis of the amplified products were performed as described in Example 7, with the exception that 0.4 μM (each) of the Staphylococcus-specific primers (SEQ ID NOs. 553 and 575) and 0.4 μM (each) of the mecA-specific primers described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and 262 in the said patent) were used in the PCR mixture. The sensitivity of the multiplex assay with 40-cycle PCR protocols was determined by using purified genomic DNA from two methicillin-resistant and five methicillin-sensitive staphylococcal strains. The detection limit was 2 to 5 copies of genomic DNA, depending on the staphylococcal species tested. The specificity of the multiplex PCR assay coupled with capture-probe hybridization was tested with two strains of methicillin-resistant S. aureus, two strains of methicillin-sensitive S. aureus and seven strains of methicillin-sensitive coagulase-negative staphylococci. The mecA-specific internal probe (SEQ ID NO. 1177) and the S. aureus-specific internal probe (SEQ ID NO. 587) described in Example 7 were able to recognize all the strains with high specificity showing a perfect correlation with susceptibility to methicillin. The sensitivity of the PCR assay coupled with capture-probe hybridization was tested with one strain of methicillin-resistant S. aureus. The detection limit was around 10 copies of genomic DNA.
Example 18
Sequencing of pbp1a, pbp2b and pbp2x Genes of Streptoccoccus pneumoniae. 
Penicillin resistance in Streptococcus pneumoniae involves the sequential alteration of up to five penicillin-binding proteins (PBPs) 1A, 1B, 2A, 2X and 2B in such a way that their affinity is greatly reduce toward the antibiotic molecule. The altered PBP genes have arisen as the result of interspecies recombination events from related streptococcal species. Among the PBPs usually found in S. pneumoniae, PBPs 1A, 2B, and 2X play the most important role in the development of penicillin resistance. Alterations in PBP 2B and 2X mediate low-level resistance to penicillin while additional alterations in PBP 1A plays a significant role in full penicillin resistance.
In order to generate a database for pbp sequences that can be used for design of primers and/or probes for the specific and ubiquitous detection of β-lactam resistance in S. pneumoniae, pbp1a, pbp2b and pbp2x DNA fragments sequenced by us or selected from public databases (GenBank and EMBL) from a variety of S. pneumoniae strains were used to design oligonucleotide primers. This database is essential for the design of specific and ubiquitous primers and/or probes for detection of β-lactam resistance in S. pneumoniae since the altered PBP 1A, PBP 2B and PBP 2X of β-lactam resistant S. pneumoniae are encoded by mosaic genes with numerous sequence variations among resistant isolates. The PCR primers were located in conserved regions of pbp genes and were able to amplify pbp1a, pbp2b, and pbp2x sequences of several strains of S. pneumoniae having various levels of resistance to penicillin and third-generation cephalosporins. Using primer pairs SEQ ID NOs. 1125 and 1126, SEQ ID NOs. 1142 and 1143, SEQ ID NOs. 1146 and 1147, it was possible to amplify and determine pbp1a sequences SEQ ID NOs. 1004-1018, 1648, 2056-2060 and 2062-2064, pbp2b sequences SEQ ID NOs. 1019-1033, and pbp2x sequences SEQ ID NOs. 1034-1048. Six other PCR primers (SEQ ID NOs. 1127-1128, 1144-1145, 1148-1149) were also designed and used to complete the sequencing of pbp1a, pbp2b and pbp2x amplification products. The described primers (SEQ ID NOs. 1125 and 1126, SEQ ID NOs. 1142 and 1143, SEQ ID NOs. 1146 and 1147, SEQ ID NOs. 1127-1128, 1144-1145, 1148-1149) represent a powerful tool for generating new pbp sequences for design of primers and/or probes for detection of β-lactam resistance in S. pneumoniae. 
Example 19
Sequencing of hexA Genes of Streptococcus Species.
The hexA sequence of S. pneumoniae described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NO. 31 in the said patent, SEQ ID NO. 1183 in the present application) allowed the design of a PCR primer (SEQ ID NO. 1182) which was used with primer Spn1401 described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NO. 156 in the said patent, SEQ ID NO. 1179 in the present application) to generate a database for hexA sequences that can be used to design primers and/or probes for the specific identification and detection of S. pneumoniae (Annex XLII). Using primers SEQ ID NO. 1179 and SEQ ID NO. 1182 (Annex XLII), it was possible to amplify and determine the hexa sequence from S. pneumoniae (4 strains) (SEQ ID NOs. 1184-1187), S. mitis (three strains) (SEQ ID NOs. 1189-1191) and S. oralis (SEQ ID NO. 1188).
Example 20
Development of Multiplex PCR Assays Coupled with Capture Probe Hybridization for the Detection and Identification of Streptococcus pneumoniae and its Penicillin Resistance Genes.
Two different assays were developed to identify S. pneumoniae and its susceptibility to penicillin.
Assay I:
Bacterial strains. The specificity of the multiplex PCR assay was verified by using a panel of ATCC (American Type Culture Collection) reference strains consisting of 33 gram-negative and 67 gram-positive bacterial species (Table 13). In addition, a total of 98 strains of S. pneumoniae, 16 strains of S. mitis and 3 strains of S. oralis from the American Type Culture Collection, the microbiology laboratory of the Centre Hospitalier Universitaire de Québec , Pavillon Centre Hospitalier de l'Université Laval (CHUL), (Step-Foy, Québec , Canada), the Laboratoire de santé publique du Québec, (Sainte-Anne-de-Bellevue, Québec, Canada), the Sunnybrook and Women's College Health Sciences Centre (Toronto, Canada), the Infectious Diseases Section, Department of Veterans Affairs Medical Center, (Houston, USA) were also tested to further validate the Streptococcus pneumoniae-specific PCR assay. The penicillin MICs (minimal inhibitory concentrations) were measured by the broth dilution method according to the recommended protocol of NCCLS.
PCR primers and internal probes. The analysis of hexA sequences from a variety of streptococcal species from the publicly available hexA sequence and from the database described in Example 19 (SEQ ID NOs. 1184-1191) allowed the selection of a PCR primer specific to S. pneumoniae, SEQ ID NO. 1181. This primer was used with the S. pneumoniae-specific primer SEQ ID NO. 1179 to generate an amplification product of 241 bp (Annex XLII). The PCR primer SEQ ID NO. 1181 is located 127 nucleotides downstream on the hexA sequence compared to the original S. pneumoniae-specific PCR primer Spn1515 described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NO. 157 in the said patent). These modifications were done to ensure the design of the S. pneumoniae-specific internal probe according to the new hexa sequences of several streptococcal species from the database described in Example 19 (SEQ ID NOs. 1184-1191).
The analysis of pbp1a sequences from S. pneumoniae strains with various levels of penicillin resistance from public databases and from the database described in Example 18 allowed the identification of amino acid substitutions Ile-459 to Met and Ser-462 to Ala that occur in isolates with high-level penicillin resistance (MICs ≧1 μg/ml), and amino acid substitutions Ser-575 to Thr, Gln-576 to Gly and Phe-577 to Tyr that are common to all penicillin-resistant isolates with MICs ≧0.25 μg/ml. As shown in Annex XXXI, PCR primer pair SEQ ID NOs. 1130 and 1131 were designed to detect high-level penicillin resistance (MICs ≧1 μg/ml), whereas PCR primer pair SEQ ID NOs. 1129 and 1131 were designed to detect intermediate- and high-level penicillin resistance (MICs ≧0.25 μg/ml).
The analysis of hexA sequences from the publicly available hexA sequence and from the database described in Example 19 allowed the design of an internal probe specific to S. pneumoniae (SEQ ID NO. 1180) (Annex XLII). The range of mismatches between the S. pneumoniae-specific 241-bp amplicon was from 2 to 5, in the middle of the 19-bp probe. The analysis of pbp1a sequences from public databases and from the database described in Example 18 allowed the design of five internal probes containing all possible mutations to detect the high-level penicillin resistance 383-bp amplicon (SEQ ID NOs. 1197, 1217-1220). Alternatively, two other internal probes (SEQ ID NOs. 2024-2025) can also be used to detect the high-level penicillin resistance 383-bp amplicon. Five internal probes containing all possible mutations to detect the 157-bp amplicon which includes intermediate- and high-level penicillin resistance were also designed (SEQ ID NOs. 1094, 1192-1193, 1214 and 1216). Design and synthesis of primers and probes, and detection of the probe hybridization were performed as described in Example 7. Annex XXXI illustrates one of the internal probe for detection of the high-level penicillin resistance 383-bp amplicon (SEQ ID NO. 1197) and one of the internal probe for detection of the intermediate- and high-level penicillin resistance 157-bp amplicon (SEQ ID NO. 1193).
PCR amplification. For all bacterial species, amplification was performed from purified genomic DNA using a PTC-200 thermocycler (MJ Research). 1 μl of genomic DNA at 0.1 ng/μl, or 1 μl of a bacterial lysate, was transferred to a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (H 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.1 μM (each) of the S. pneumoniae-specific primers SEQ ID NO. 1179 and SEQ ID NO. 1181, 0.2 μM of primer SEQ ID NO. 1129, 0.7 μM of primer SEQ ID NO. 1131, and 0.6 μM of primer SEQ ID NO. 1130, 0.05 mM bovine serum albumin (BSA), and 0.5 U Taq polymerase (Promega) coupled with TaqStartTM antibody. In order to generate Digoxigenin (DIG)-labeled amplicons for capture probe hybridization, 0.1×PCR DIG labeling four deoxynucleoside triphosphates mix (Boehringer Mannheim GmbH) was used for amplification.
For determination of the sensitivity of the PCR assays, 10-fold dilutions of purified genomic DNA were used to determine the minimal number of genome copies which can be detected.
Capture probe hybridization. The DIG-labeled amplicons were hybridized to the capture probes bound to 96-well plates. The plates were incubated with anti-DIG-alkaline phosphatase and the chemiluminescence was measured by using a luminometer (MLX, Dynex Technologies Inc.) after incubation with CSPD and recorded as Relative Light Unit (RLU). The RLU ratio of tested sample with and without captures probes was then calculated. A ratio ≧2.0 was defined as a positive hybridization signal. All reactions were performed in duplicate.
Results
Amplifications with the multiplex PCR assay. The specificity of the assay was assessed by performing 40-cycle PCR amplifications with the panel of gram-positive (67 species from 12 genera) and gram-negative (33 species from 17 genera) bacterial species listed in Table 13. All bacterial species tested other than S. pneumoniae were negative except S. mitis and S. oralis. Ubiquity tests were performed using a collection of 98 S. pneumoniae strains including high-level penicillin resistance (n=53), intermediate resistance (n=12) and sensitive (n=33) strains. There was a perfect correlation between PCR and standard susceptibility testing for 33 penicillin-sensitive isolates. Among 12 S. pneumoniae isolates with intermediate penicillin resistance based on susceptibility testing, 11 had intermediate resistance based on PCR, but one S. pneumoniae isolate with penicillin MIC of 0.25 μg/ml showed a high-level penicillin resistance based on genotyping. Among 53 isolates with high-level penicillin resistance based on susceptibility testing, 51 had high-level penicillin resistance based on PCR but two isolates with penicillin MIC >1 μg/ml showed an intermediate penicillin resistance based on genotyping. In general, there was a good correlation between the genotype and classical culture method for bacterial identification and susceptibility testing.
The sensitivity of the S. pneumoniae-specific assay with 40-cycle PCR protocols was determined by using purified genomic DNA from 9 isolates of S. pneumoniae. The detection limit was around 10 copies of genomic DNA for all of them.
Post-PCR hybridization with internal probes. The specificity of the multiplex PCR assay coupled with capture-probe hybridization was tested with 98 strains of S. pneumoniae, 16 strains of S. mitis and 3 strains of S. oralis. The internal probe specific to S. pneumoniae (SEQ ID NO. 1180) detected all 98 S. pneumoniae strains but did not hybridize to the S. mitis and S. oralis amplicons. The five internal probes specific to the high-level resistance amplicon (SEQ ID NOs. 1197, 1217-1220) detected all amplification patterns corresponding to high-level resistance. The two S. pneumoniae trains with penicillin MIC >1 μg/ml that showed an intermediate penicillin resistance based on PCR amplification were also intermediate resistance based on probe hybridization. Similarly, among 12 strains with intermediate-penicillin resistance based on susceptibility testing, 11 showed intermediate-penicillin resistance based on hybridization with the five internal probes specific to the intermediate and high-level resistance amplicon (SEQ ID NOs. 1094, 1192-1193, 1214 and 1216). The strain described above having a penicillin MIC of 0.25 μg/ml which was high-level penicillin resistance based on PCR amplification was also high-level resistance based on probe hybridization. In summary, the combination of the multiplex PCR and hybridization assays results in a highly specific test for the detection of penicillin-resistant Streptococcus pneumoniae. 
Assay II:
Bacterial strains. The specificity of the multiplex PCR assay was verified by using the same strains as those used for the development of Assay I. The penicillin MICs (minimal inhibitory concentrations) were measured by the broth dilution method according to the recommended protocol of NCCLS.
PCR primers and internal probes. The analysis of pbp1a sequences from S. pneumoniae strains with various levels of penicillin resistance from public databases and from the database described in Example 18 allowed the design of two primers located in the constant region of pbp1a. PCR primer pair (SEQ ID NOs. 2015 and 2016) was designed to amplify a 888-bp variable region of pbp1a from all S. pneumoniae strains. A series of internal probes were designed for identification of the pbp1a mutations associated with penicillin resistance in S. pneumoniae. For detection of high-level penicillin resistance (MICs ≧1 μg/ml), three internal probes were designed (SEQ ID NOs. 2017-2019). Alternatively, ten other internal probes were designed that can also be used for detection of high-level resistance within the 888-bp pbp1a amplicon: (1) three internal probes for identification of the amino acid substitutions Thr-371 to Ser or Ala within the motif S370TMK (SEQ ID NOs. 2031-2033); (2) two internal probes for detection of the amino acid substitutions Ile-459 to Met and Ser-462 to Ala near the motif S428RN (SEQ ID NOs. 1135 and 2026); (3) two internal probes for identification of the amino acid substitutions Asn-443 to Asp (SEQ ID NOs. 1134 and 2027); and (4) three internal probes for detection of all sequence variations within another region (SEQ ID NOs. 2028-2030). For detection of high-level and intermediate penicillin resistance (MICs ≧0.25 μg/ml), four internal probes were designed (SEQ ID NOs. 2020-2023). Alternatively, six other internal probes were designed for detection of the four consecutive amino acid substitutions T574SQF to AS74TGY near the motif K557TG (SEQ ID NOs. 2034-2039) that can also be used for detection of intermediate- and high-level resistance within the 888-bp pbp1a amplicon.
PCR amplification. For all bacterial species, amplification was performed from purified genomic DNA using a PTC-200 thermocycler (MJ Research). 1 μl of genomic DNA at 0.1 ng/μl, or 1 μl of a bacterial lysate, was transferred to a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.08 μM (each) of the S. pneumoniae-specific primers SEQ ID NO. 1179 and SEQ ID NO. 1181, 0.4 μM of the pbp1a-specific primer SEQ ID NO. 2015, 1.2 μM of pbp1a-specific primer. SEQ ID NO. 2016, 0.05 mM bovine serum albumin (BSA), and 0.5 U Taq polymerase (Promega) coupled with TaqStartTM antibody. In order to generate Digoxigenin (DIG)-labeled amplicons for capture probe hybridization, 0.1×PCR DIG labeling four deoxynucleoside triphosphates mix (Boehringer Mannheim GmbH) was used for amplification.
For determination of the sensitivities of the PCR assays, 10-fold dilutions of purified genomic DNA were used to determine the minimal number of genome copies which can be detected.
Capture probe hybridization. The DIG-labeled amplicons were hybridized to the capture probes bound to 96-well plates as described for Assay I.
Results
Amplifications with the multiplex PCR assay. The specificity of the assay was assessed by performing 40-cycle PCR amplifications with the panel of gram-positive (67 species from 12 genera) and gram-negative (33 species from 17 genera) bacterial species listed in Table 13. All bacterial species tested other than S. pneumoniae were negative except S. mitis and S. oralis. Ubiquity tests were performed using a collection of 98 S. pneumoniae strains including high-level penicillin resistance (n=53), intermediate resistance (n=12) and sensitive (n=33) strains. All the above S. pneumoniae strains produced the 888-bp amplicon corresponding to pbp1a and the 241-bp fragment corresponding to hexA.
The sensitivity of the S. pneumoniae-specific assay with 40-cycle PCR protocols was determined by using purified genomic DNA from 9 isolates of S. pneumoniae. The detection limit was around 10 copies of genomic DNA for all of them.
Post-PCR hybridization with internal probes. The specificity of the multiplex PCR assay coupled with capture-probe hybridization was tested with 98 strains of S. pneumoniae, 16 strains of S. mitis and 3 strains of S. oralis. The internal probe specific to S. pneumoniae (SEQ ID NO. 1180) detected all 98 S. pneumoniae strains but did not hybridize to the S. mitis and S. oralis amplicons. The three internal probes (SEQ ID NOs 2017-2019) specific to high-level resistance detected all the 43 strains with high-level penicillin resistance based on susceptibility testing. Among 12 isolates with intermediate-penicillin resistance based on susceptibility testing, 11 showed intermediate-penicillin resistance based on hybridization with 4 internal probes (SEQ ID NOs. 2020-2023) and one strain having penicillin MIC of 0.25 μg/ml was misclassified as high-level penicillin resistance. In summary, the combination of the multiplex PCR and hybridization assays results in a highly specific test for the detection of penicillin-resistant Streptococcus pneumoniae. 
Example 21
Sequencing of the Vancomycin Resistance vanA, vanC1, vanC2 and vanC3 Genes.
The publicly available sequences of the vanH-vanA-vanX-vanY locus of transposon Tn1546 from E. faecalis, vanC1 sequence from one strain of E. gallinarum, vanC2 and vanC3 sequences from a variety of E. casseliflavus and E. flavescens strains, respectively, allowed the design of PCR primers able to amplify the vanA, vanC1, vanC2 and vanC3 sequences of several Enterococcus species. Using primer pairs van6877 and van9106 (SEQ ID NOs. 1150 and 1155), vanC1-122 and vanC1-1315 (SEQ ID NOs. 1110 and 1109), and vanC2C3-1 and vanC2C3-1064 (SEQ ID NOs. 1108 and 1107), it was possible to amplify and determine vanA sequences SEQ ID NOs. 1049-1057, vanC1 sequences SEQ ID NOs. 1058-1059, vanC2 sequences SEQ ID NOs. 1060-1063 and vanC3 sequences SEQ ID NOs. 1064-1066, respectively. Four other PCR primers (SEQ ID NOs. 1151-1154) were also designed and used to complete the sequencing of vanA amplification products.
Example 22
Development of a PCR Assay for the Detection and Identification of Enterococci at Genus and Species Levels and its Associated Resistance Genes vanA and vanB.
The comparison of vanA and vanB sequences revealed conserved regions allowing the design of PCR primers specific to both vanA and vanB sequences (Annex XXXVIII). The PCR primer pair vanAB459 and vanAB830R (SEQ ID NOs. 1112 and 1111) was used in multiplex with the Enterococcus-specific primers Encg313dF and Encg599c (SEQ ID NOs. 1137 and 1136) described in Example 11. Sequence alignment analysis of vanA and vanB sequences revealed regions suitable for the design of internal probes specific to vanA (SEQ ID NO. 1170) and vanB (SEQ ID NO. 1171). PCR amplification and agarose gel electropheresis of the amplified products were performed as described in Example 11. The optimal cycling conditions for maximum sensitivity and specificity were found to be 3 min. at 94° C., followed by forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 62° C., plus a terminal extension at 72° C. for 2 minutes. The specificity of the multiplex assay with 40-cycle PCR was verified by using 0.1 nanogram of purified genomic DNA from a panel of bacteria listed in Table 10. The sensitivity of the multiplex assay with 40-cycle PCR was verified with three strains of E. casseliflavus, eight strains of E. gallinarum, two strains of E. flavescens, two vancomycin-resistant strains of E. faecalis and one vancomycin-sensitive strain of E. faecalis, three vancomycin-resistant strains of E. faecium, one vancomycin-sensitive strain of E. faecium and one strain of each of the other enterococcal species listed in Table 10. The detection limit was 1 to 10 copies of genomic DNA, depending on the enterococcal species tested. The vanA- and vanB-specific internal probes (SEQ ID NOs. 1170 and 1171), as well as the E. faecalis- and E. faecium-specific internal probes (SEQ ID NOs. 1174 and 602) and the internal probe specific to the group including E. casseliflavus, E. gallinarum and E. flavescens (SEQ ID NO. 1122) described in Example 11, were able to recognize vancomycin-resistant enterococcal species with high sensitivity, specificity and ubiquity showing a perfect correlation between the genotypic and phenotypic analysis.
The format of the assay is not limited to the one described above. A person skilled in the art could adapt the assay for different formats such as PCR with real-time detection using molecular beacon probes. Molecular beacon probes designed to be used in this assay include, but are not limited to, SEQ ID NO. 1236 for the detection of E. faecalis, SEQ ID NO. 1235 for the detection of E. faecium, SEQ ID NO. 1240 for the detection of vanA, and SEQ ID NO. 1241 for the detection of vanB.
Example 23
Development of a Multiplex PCR Assay for Detection and Identification of Vancomycin-Resistant Enterococcus faecalis, Enterococcus faecium and the Group Including Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens. 
The analysis of vanA and vanB sequences revealed conserved regions allowing design of a PCR primer pair (SEQ ID NOs. 1089 and 1090) specific to vanA sequences (Annex XXVIII) and a PCR primer pair (SEQ ID NOs. 1095 and 1096) specific to vanB sequences (Annex XXIX). The vanA-specific PCR primer pair (SEQ ID NOs. 1089 and 1090) was used in multiplex with the vanB-specific PCR primer pair described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 1095 and 1096 in the present patent and SEQ ID NOs. 231 and 232 in the said patent). The comparison of vanC1, vanC2 and vanC3 sequences revealed conserved regions allowing design of PCR primers (SEQ ID NOs. 1101 and 1102) able to generate a 158-bp amplicon specific to the group including E. gallinarum, E. casseliflavus and E. flavescens (Annex XXX). The vanC-specific PCR primer pair (SEQ ID NOs. 1101 and 1102) was used in multiplex with the E. faecalis-specific PCR primer pair described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 40 and 41 in the said patent) and with the E. faecium-specific PCR primer pair described in our patent publication WO98/20157 (SEQ ID NOs. 1 and 2 in the said publication). For both multiplexes, the optimal cycling conditions for maximum sensitivity and specificity were found to be 3 min. at 94° C., followed by forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 58° C., plus a terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. The vanA-specific PCR primer pair (SEQ ID NOs. 1089 and 1090), the vanB-specific primer pair (SEQ ID NOs. 1095 and 1096) and the vanC-specific primer pair (SEQ ID NOs. 1101 and 1102) were tested for their specificity by using 0.1 nanogram of purified genomic DNA from a panel of 5 vancomycin-sensitive Enterococcus species, 3 vancomycin-resistant Enterococcus species, 13 other gram-positive bacteria and one gram-negative bacterium. Specificity tests were performed with the E. faecium-specific PCR primer pair described in our patent publication WO98/20157 (SEQ ID NOs. 1 and 2 in the said publication) and with the E. faecalis-specific PCR primer pair described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 40 and 41 in the said patent) on a panel of 37 gram-positive bacterial species. All Enterococcus strains were amplified with high specificity showing a perfect correlation between the genotypic and phenotypic analysis. The sensitivity of the assays was determined for several strains of E. gallinarum, E. casseliflavus, E. flavescens and vancomycin-resistant E. faecalis and E. faecium. Using each of the E. faecalis- and E. faecium-specific PCR primer pairs as well as vanA-, vanB- and vanC-specific PCR primers used alone or in multiplex as described above, the sensitivity ranged from 1 to 10 copies of genomic DNA.
The format of the assay is not limited to the one described above. A person skilled in the art could adapt the assay for different formats such as PCR with real-time detection using molecular beacon probes. Molecular beacon probes designed to be used in this assay include, but are not limited to, SEQ ID NO. 1238 for the detection of E. faecalis, SEQ ID NO. 1237 for the detection of E. faecium, SEQ ID NO. 1239 for the detection of vanA, and SEQ ID NO. 1241 for the detection of vanB.
Alternatively, another PCR assay was developed for the detection of vancomycin-resistant E. faecium and vancomycin-resistant E. faecalis. This assay included two multiplex: (1) the first multiplex contained the vanA-specific primer pair (SEQ ID NOs. 1090-1091) and the vanB-specific PCR primer pair described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 1095 and 1096 in the present patent and SEQ ID NOs. 231 and 232 in the said patent), and (2) the second multiplex contained the E. faecalis-specific PCR primer pair described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 40 and 41 in the said patent) and the E. faecium-specific PCR primer pair described in our patent publication WO98/20157 (SEQ ID NOs. 1 and 2 in the said publication). For both multiplexes, the optimal cycling conditions for maximum sensitivity and specificity were found to be 3 min. at 94° C., followed by forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 58° C., plus a terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. The two multiplexes were tested for their specificity by using 0.1 nanogram of purified genomic DNA from a panel of two vancomycin-sensitive E. faecalis strains, two vancomycin-resistant E. faecalis strains, two vancomycin-sensitive E. faecium strains, two vancomycin-resistant E. faecium strains, 16 other enterococcal species and 31 other gram-positive bacterial species. All the E. faecium and E. faecalis strains were amplified with high specificity showing a perfect correlation between the genotypic analysis and the susceptibility to glycopeptide antibiotics (vancomycin and teicoplanin). The sensitivity of the assay was determined for two vancomycin-resistant E. faecalis strains and two vancomycin-resistant E. faecium strains. The detection limit was 5 copies of genomic DNA for all the strains.
This multiplex PCR assay was coupled with capture-probe hybridization. Four internal probes were designed: one specific to the vanA amplicon (SEQ ID NO. 2292), one specific to the vanB amplicon (SEQ ID NO. 2294), one specific to the E. faecalis amplicon (SEQ ID NO. 2291) and one specific to the E. faecium amplicon (SEQ ID NO. 2287). Each of the internal probes detected their specific amplicons with high specificity and sensitivity.
Example 24
Universal Amplification Involving the EF-G (fusA) Subdivision of tuf Sequences.
As shown in FIG. 3, primers SEQ ID NOs. 1228 and 1229 were designed to amplify the region between the end of fusA and the beginning of tuf genes in the str operon. Genomic DNAs from a panel of 35 strains were tested for PCR amplification with those primers. In the initial experiment, the following strains showed a positive result: Abiotrophia adiacens ATCC 49175, Abiotrophia defectiva ATCC 49176, Bacillus subtilis ATCC 27370, Closridium difficile ATCC 9689, Enterococcus avium ATCC 14025, Enterococcus casseliflavus ATCC 25788, Enterococcus cecorum ATCC 43198, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 19434, Enterococcus flavescens ATCC 49996, Enterococcus gallinarum ATCC 49573, Enterococcus solitarius ATCC 49428, Escherichia coli ATCC 11775, Haemophilus influenzae ATCC 9006, Lactobacillus acidophilus ATCC 4356, Peptococcus niger ATCC 27731, Proteus mirabilis ATCC 25933, Staphylococcus aureus ATCC 43300, Staphylococcus auricularis ATCC 33753, Staphylococcus capitis ATCC 27840, Staphylococcus epidemidis ATCC 14990, Staphylococcus haemolyticus ATCC 29970, Staphylococcus hominis ATCC 27844, Staphylococcus lugdunensis ATCC 43809, Staphylococcus saprophyticus ATCC 15305, Staphylococcus simulans ATCC 27848, and Staphylococcus warneri ATCC 27836. This primer pair could amplify additional bacterial species; however, there was no amplification for some species, suggesting that the PCR cycling conditions could be optimized or the primers modified. For example, SEQ ID NO. 1227 was designed to amplify a broader range of species.
In addition to other possible primer combinations to amplify the region covering fusA and tuf, FIG. 3 illustrates the positions of amplification primers SEQ ID NOs. 1221-1227 which could be used for universal amplification of fusA segments. All of the above mentioned primers (SEQ ID NOs. 1221-1229) could be useful for the universal and/or the specific detection of bacteria.
Moreover, different combinations of primers SEQ ID NOs. 1221-1229, sometimes in combination with tuf sequencing primer SEQ ID NO. 697, were used to sequence portions of the str operon, including the intergenic region. In this manner, the following sequences were generated: SEQ ID NOs. 1518-1526, 1578-1580, 1786-1821, 1822-1834, 1838-1843, 2184, 2187, 2188, 2214-2249, and 2255-2269.
Example 25
DNA Fragment Isolation from Staphylococcus saprophyticus by Arbitrarily Primed PCR.
DNA sequences of unknown coding potential for the species-specific detection and identification of Staphylococcus saprophyticus were obtained by the method of arbitrarily primed PCR (AP-PCR).
AP-PCR is a method which can be used to generate specific DNA probes for microorganisms (Fani et al., 1993, Molecular Ecology 2:243-250). A description of the AP-PCR protocol used to isolate a species-specific genomic DNA fragment from Staphylococcus saprophyticus follows. Twenty different oligonucleotide primers of 10 nucleotides in length (all included in the AP-PCR kit OPAD (Operon Technologies, Inc., Alameda, Calif.)) were tested systematically with DNAs from 5 bacterial strains of Staphylococcus saprophyticus as well as with bacterial strains of 27 other staphylococcal (non-S. saprophyticus) species. For all bacterial species, amplification was performed directly from one μL (0.1 ng/μL) of purified genomic DNA. The 25 μL PCR reaction mixture contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 1.2 μM of only one of the 20 different AP-PCR primers OPAD, 200 μM of each of the four dNTPs, 0.5 U of Taq DNA polymerase (Promega Corp., Madison, Wis.) coupled with TaqStartTM antibody (Clontech Laboratories Inc., Palo Alto, Calif.). PCR reactions were subjected to cycling using a MJ Research PTC-200 thermal cycler as follows: 3 min at 96° C. followed by 42 cycles of 1 min at 94° C. for the denaturation step, 1 min at 31° C. for the annealing step and 2 min at 72° C. for the extension step. A final extension step of 7 min at 72° C. followed the 42 cycles to ensure complete extension of PCR products. Subsequently, twenty microliters of the PCR-amplified mixture were resolved by electrophoresis on a 1.5% agarose gel containing 0.25 μg/ml of ethidium bromide. The size of the amplification products was estimated by comparison with a 50-bp molecular weight ladder.
Amplification patterns specific for Staphylococcus saprophyticus were observed with the AP-PCR primer OPAD-16 (sequence: 5′-AACGGGCGTC-3′). Amplification with this primer consistently showed a band corresponding to a DNA fragment of approximately 380 bp for all Staphylococcus saprophyticus strains tested but not for any of the other staphylococcal species tested.
The band corresponding to the 380 bp amplicon, specific and ubiquitous for S. saprophyticus based on AP-PCR, was excised from the agarose gel and purified using the QIAquickTM gel extraction kit (QIAGEN Inc.). The gel-purified DNA fragment was cloned into the T/A cloning site of the pCR 2.1TM plasmid vector (Invitrogen Inc.) using T4 DNA ligase (New England BioLabs). Recombinant plasmids were transformed into E. coli DH5α competent cells using standard procedures. All reactions were performed according to the manufacturer's instructions. Plasmid DNA isolation was done by the method of Birnboim and Doly (Nucleic Acid Res., 1979, 7:1513-1523) for small-scale preparations. All plasmid DNA preparations were digested with the EcoRI restriction endonuclease to ensure the presence of the approximately 380 bp AP-PCR insert into the plasmid. Subsequently, a large-scale and highly purified plasmid DNA preparation was performed from two selected clones shown to carry the AP-PCR insert by using the QIAGEN plasmid purification kit (midi format). These large-scale plasmid preparations were used for automated DNA sequencing.
The 380 bp nucleotide sequence was determined for three strains of S. saprophyticus (SEQ ID NOs. 74, 1093, and 1198). Both strands of the AP-PCR insert from the two selected clones were sequenced by the dideoxynucleotide chain termination sequencing method with SP6 and T7 sequencing primers by using the Applied Biosystems automated DNA sequencer (model 373A) with their PRISMTM Sequenase® Terminator Double-stranded DNA Sequencing Kit (Applied Biosystems, Foster City, Calif.).
Optimal species-specific amplification primers (SEQ ID NOs. 1208 and 1209) have been selected from the sequenced AP-PCR Staphylococcus saprophyticus DNA fragments with the help of the primer analysis software OligoTM 5.0 (National BioSciences Inc.). The selected primers were tested in PCR assays to verify their specificity and ubiquity. Data obtained with DNA preparations from reference ATCC strains of 49 gram-positive and 31 gram-negative bacterial species, including 28 different staphylococcal species, indicate that the selected primer pairs are specific for Staphylococcus saprophyticus since no amplification signal has been observed with DNAs from the other staphylococcal or bacterial species tested. This assay was able to amplify efficiently DNA from all 60 strains of S. saprophyticus from various origins tested. The sensitivity level achieved for three S. saprophyticus reference ATCC strains was around 6 genome copies.
Example 26
Sequencing of Prokaryotic tuf Gene Fragments.
The comparison of publicly available tuf sequences from a variety of bacterial species revealed conserved regions, allowing the design of PCR primers able to amplify tuf sequences from a wide range of bacterial species. Using primer pair SEQ ID NOs. 664 and 697, it was possible to amplify and determine tuf sequences SEQ ID NOs.: 1-73, 75-241, 607-618, 621, 662, 675, 717-736, 868-888, 932, 967-989, 992, 1002, 1572-1575, 1662-1663, 1715-1733, 1835-1837, 1877-1878, 1880-1881, 2183, 2185, 2200, 2201, and 2270-2272.
Example 27
Sequencing of Procaryotic recA Gene Fragments.
The comparison of publicly available recA sequences from a variety of bacterial species revealed conserved regions, allowing the design of PCR primers able to amplify recA sequences from a wide range of bacterial species. Using primer pairs SEQ ID NOs. 921-922 and 1605-1606, it was possible to amplify and determine recA sequences SEQ ID NOs.: 990-991, 1003, 1288-1289, 1714, 1756-1763, 1866-1873 and 2202-2212.
Example 28
Specific Detection and Identification of Escherichia coli/Shigella sp. Using tuf Sequences.
The analysis of tuf sequences from a variety of bacterial species allowed the selection of PCR primers (SEQ ID NOs. 1661 and 1665) and of an internal probe (SEQ ID NO. 2168) specific to Escherichia coli/Shigella sp. The strategy used to design the PCR primers was based on the analysis of a multiple sequence alignment of various tuf sequences. The multiple sequence alignment included the tuf sequences of Escherichia coli/Shigella sp. as well as tuf sequences from other species and bacterial genera, especially representatives of closely related species. A careful analysis of this alignment allowed the selection of oligonucleotide sequences which are conserved within the target species but which discriminate sequences from other species, especially from the closely related species, thereby permitting the species-specific and ubiquitous detection and identification of the target bacterial species.
The chosen primer pair, oligos SEQ ID NOs. 1661 and 1665, gives an amplification product of 219 bp. Standard PCR was carried out using 0.4 μM of each primer, 2.5 mM MgCl2, BSA 0.05 mM, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, dNTPs 0.2 mM (Pharmacia), 0.5 U Taq DNA polymerase (Promega) coupled with TaqStartTM antibody (Clontech Laboratories Inc.), 1 μl of genomic DNA sample in a final volume of 20 μl using a PTC-200 thermocycler (MJ Research). The optimal cycling conditions for maximum sensitivity and specificity were 3 minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 60° C., followed by terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. Visualization of the PCR products was made under UV at 254 nm.
Specificity of the assay was tested by adding to the PCR reactions 0.1 ng of genomic DNA from each of the following bacterial species: Escherichia coli (7 strains), Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Salmonella typhimyurium, Salmonella typhi, Salmonella enteritidis, Tatumella ptyseos, Klebsiella pneumoniae (2 strains), Enterobacter aerogenes, Citrobacter farmeri, Campylobacter jejuni, Serratia marcescens. Amplification was observed only for the Escherichia coli and Shigella sp. strains listed and Escherichia fergusonii. The sensitivity of the assay with 40-cycle PCR was verified with one strain of E. coli and three strains of Shigella sp. The detection limit for E. coli and Shigella sp. was 1 to 10 copies of genomic DNA, depending on the strains tested.
Example 29
Specific Detection and Identification of Klebsiella pneumoniae Using atpD Sequences.
The analysis of atpD sequences from a variety of bacterial species allowed the selection of PCR primers specific to K. pneumoniae. The primer design strategy is similar to the strategy described in Example 28 except that atpD sequences were used in the alignment.
Two K. pneumoniae-specific primers were selected, (SEQ ID NOs. 1331 and 1332) which give an amplification product of 115 bp. Standard PCR was carried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM of each primer as described in Example 28. The optimal cycling conditions for maximum sensitivity and specificity were as follow: three minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 55° C., followed by terminal extension at 72° C. for 2 minutes.
Specificity of the assay was tested by adding to the PCR reactions 0.1 ng of genomic DNA from each of the following bacterial species: Klebsiella pneumoniae (2 strains), Klebsiella ornitholytica, Klebsiella oxytoca (2 strains), Klebsiella planticola, Klebsiella terrigena, Citrobacter freundii, Escherichia coli, Salmonella cholerasuis typhi, Serratia marcescens, Enterobacter aerogenes, Proteus vulgaris, Kluyvera ascorbata, Kluyvera georgiana, Kluyvera cryocrescens and Yersinia enterolitica. Amplification was detected for the two K. pneumoniae strains, K. planticola, K. terrigena and the three Kluyvera species tested. Analysis of the multiple alignment sequence of the atpD gene allowed the design of an internal probe SEQ ID NO. 2167 which can discrimate Klebsiella pneumoniae from other Klebsiella sp. and Kluyvera sp. The sensitivity of the assay with 40-cycle PCR was verified with one strain of K. pneumoniae. The detection limit for K. pneumoniae was around 10 copies of genomic DNA.
Example 30
Specific Detection and Identification of Acinetobacter baumannii Using atpD Sequences.
The analysis of atpD sequences from a variety of bacterial species allowed the selection of PCR primers specific to Acinetobacter baumannii. The primer design strategy is similar to the strategy described in Example 28.
Two A. baumannii-specific primers were selected, SEQ ID NOs. 1690 and 1691, which give an amplification product of 233 bp. Standard PCR was carried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM of each primer as described in Example 28. The optimal cycling conditions for maximum sensitivity and specificity were as follow: three minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 60° C., followed by terminal extension at 72° C. for 2 minutes.
Specificity of the assay was tested by adding to the PCR reactions 0.1 ng of genomic DNA from each of the following bacterial species: Acinetobacter baumannii (3 strains), Acinetobacter anitratus, Acinetobacter lwoffi, Serratia marcescens, Enterobacter cloacae, Enterococcus faecalis, Pseudomonas aeruginosa, Psychrobacter phenylpyruvicus, Neisseria gonorrheoae, Haemophilus haemoliticus, Yersinia enterolitica, Proteus vulgaris, Eikenella corrodens, Escherichia coli. Amplification was detected only for A. baumannii, A. anitratus and A. lwoffi. The sensitivity of the assay with 40-cycle PCR was verified with two strains of A. baumannii. The detection limit for the two A. baumannii strains tested was 5 copies of genomic DNA. Analysis of the multiple alignment sequence of the atpD gene allowed the design of a A. baumannii-specific internal probe (SEQ ID NO. 2169).
Example 31
Specific Detection and Identification of Neisseria gonorrhoeae Using tuf Sequences.
The analysis of tuf sequences from a variety of bacterial species allowed the selection of PCR primers specific to Neisseria gonorrhoeae. The primer design strategy is similar to the strategy described in Example 28.
Two N. gonorrhoeae-specific primers were selected, SEQ ID NOs. 551 and 552, which give an amplification product of 139 bp. PCR amplification was carried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM of each primer as described in Example 28. The optimal cycling conditions for maximum sensitivity and specificity were as follow: three minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 65° C., followed by terminal extension at 72° C. for 2 minutes.
Specificity of the assay was tested by adding into the PCR reactions, 0.1 ng of genomic DNA from each of the following bacterial species: Neisseria gonorrhoeae (19 strains), Neisseria meningitidis (2 strains), Neisseria lactamica, Neisseria flavescens, Neisseria animalis, Neisseria canis, Neisseria cuniculi, Neisseria elongata, Neisseria mucosa, Neisseria polysaccharea, Neisseria sicca, Neisseria subflava, Neisseria weaveri. Amplification was detected only for N. gonorrhoeae, N. sicca and N. polysaccharea. The sensitivity of the assay with 40-cycle PCR was verified with two strains of N. gonorrhoeae. The detection limit for the N. gonorrhoeae strains tested was 5 copies of genomic DNA. Analysis of the multiple alignment sequence of the tuf gene allowed the design of an internal probe, SEQ ID NO. 2166, which can discriminate N. gonorrhoeae from N. sicca and N. polysaccharea. 
Example 32
Sequencing of Bacterial gyrA and parC Gene Fragments. Sequencing of Bacterial gyrA and parC Fragments.
One of the major mechanism of resistance to quinolone in various bacterial species is mediated by target changes (DNA gyrase and/or topoisomerase IV). These enzymes control DNA topology and are vital for chromosome function and replication. Each of these enzymes is a tetramer composed of two subunits: GyrA and GyrB forming A2B2 complex in DNA gyrase; and ParC and ParE forming and C2E2 complex in DNA topoisomerase IV. It has been shown that they are hotspots, called the quinolone-resistance-determining region (QRDR) for mutations within gyrA that encodes for the GyrA subunit of DNA gyrase and within parC that encodes the parC subunit of topoisomerase IV.
In order to generate a database for gyrA and parC sequences that can be used for design of primers and/or probes for the specific detection of quinolone resistance in various bacterial species, gyrA and parC DNA fragments selected from public database (GenBanK and EMBL) from a variety of bacterial species were used to design oligonucleotide primers.
Using primer pair SEQ ID NOs. 1297 and 1298, it was possible to amplify and determine gyrA sequences from Klebsiella oxytoca (SEQ ID NO. 1764), Klebsiella pneumoniae subsp. ozaneae (SEQ ID NO. 1765), Klebsiella planticola (SEQ ID NO. 1766), Klebsiella pneumoniae (SEQ ID NO. 1767), Klebsiella pneumoniae subsp. pneumoniae (two strains) (SEQ ID NOs. 1768-1769), Klebsiella pneumoniae subsp. rhinoscleromatis (SEQ ID NO. 1770), Klebsiella terrigena (SEQ ID NO. 1771), Kluyvera ascorbata (SEQ ID NO. 2013), Kluyvera georgiana (SEQ ID NO. 2014) and Escherichia coli (4 strains) (SEQ ID NOs. 2277-2280). Using primer pair SEQ ID NOs. 1291 and 1292, it was possible to amplify and determine gyrA sequences from Legionella pneumophila subsp. pneumophila (SEQ ID NO. 1772), Proteus mirabilis (SEQ ID NO. 1773), Providencia rettgeri (SEQ ID NO. 1774), Proteus vulgaris (SEQ ID NO. 1775) and Yersinia enterolitica (SEQ ID NO. 1776). Using primer pair SEQ ID NOs. 1340 and 1341, it was possible to amplify and determine gyrA sequence from Staphylococcus aureus (SEQ ID NO. 1255).
Using primers SEQ ID NOs. 1318 and 1319, it was possible to amplify and determine parC sequences from K. oxytoca (two strains) (SEQ ID NOs. 1777-1778), Klebsiella pneumoniae subsp. ozaenae (SEQ ID NO. 1779), Klebsiella planticola (SEQ ID NO. 1780), Klebsiella pneumoniae (SEQ ID NO. 1781), Klebsiella pneumoniae subsp. pneumoniae (two strains) (SEQ ID NOs. 1782-1783), Klebsiella pneumoniae subsp. rhinoscleromatis (SEQ ID NO. 1784) and Klebsiella terrigena (SEQ ID NO. 1785).
Example 33
Development of a PCR Assay for the Specific Detection and Identification of Staphylococcus aureus and its Quinolone Resistance Genes gyrA and parC.
The analysis of gyrA and parC sequences from a variety of bacterial species revealed conserved regions allowing the design of PCR primers specific to the quinolone-resistance-determining region (QRDR) of gyrA and parC from Staphylococcus aureus. PCR primer pair SEQ ID NOs. 1340 and 1341 was designed to amplify the gyrA sequence of S. aureus, whereas PCR primer pair SEQ ID NOs. 1342 and 1343 was designed to amplify S. aureus parC. The comparison of gyrA and parC sequences from S. aureus strains with various levels of quinolone resistance allowed the identification of amino acid substitutions Ser-84 to Leu, Glu-88 to Gly or Lys in the GyrA subunit of DNA gyrase encoded by gyrA and amino acid changes Ser-80 to Phe or Tyr and Ala-116 to Glu in the ParC subunit of topoisomerase IV encoded by parC. These amino acid substitutions in GyrA and ParC subunits occur in isolates with intermediate- or high-level quinolone resistance. Internal probes for the specific detection of wild-type S. aureus gyrA (SEQ ID NO. 1940) and wild-type S. aureus parC (SEQ ID NO. 1941) as well as internal probes for the specific detection of each of the gyrA (SEQ ID NOs. 1333-1335) and parC mutations identified in quinolone-resistant S. aureus (SEQ ID NOs. 1336-1339) were designed.
The gyrA- and parC-specific primer pairs (SEQ ID NOs. 1340-1341 and SEQ ID NOs. 1342-1343) were used in multiplex. PCR amplification was carried out on PTC-200 thermocyclers (MJ Research) using 0.3, 0.3, 0.6 and 0.6 μM of each primers, respectively, as described in Example 28. The optimal cycling conditions for maximum sensitivity and specificity were 3 minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 62° C., followed by terminal extension at 72° C. for 2 minutes. Detection of the PCR products was made by electrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidium bromide. The specificity of the multiplex assay with 40-cycle PCR was verified by using 0.1 ng of purified genomic DNA from a panel of gram-positive bacteria. The list included the following: Abiotrophia adiacens, Abiotrophia defectiva, Bacillus cereus, Bacillus mycoides, Enterococcus faecalis (2 strains), Enterococcus flavescens, Gemella morbillorum, Lactococcus lactis, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus (5 strains), Staphylococcus auricalis, Staphylococcus capitis subsp. urealyticus, Staphylococcus carnosus, Staphylococcus chromogenes, Staphylococcus epidermidis (3 strains), Staphylococcus gallinarum, Staphylococcus haemolyticus (2 strains), Staphylococcus hominis, Staphylococcus hominis subsp hominis, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus saccharolyticus, Staphylococcus saprophyticus (3 strains), Staphylococcus simulans, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus pneumoniae. Strong amplification of both gyrA and parC genes was only detected for the S. aureus strains tested. The sensitivity of the multiplex assay with 40-cycle PCR was verified with one quinolone-sensitive and four quinolone-resistant strains of S. aureus. The detection limit was 2 to 10 copies of genomic DNA, depending on the strains tested.
Detection of the hybridization with the internal probes was performed as described in Example 7. The internal probes specific to wild-type gyrA and parC of S. aureus and to the gyrA and parC variants of S. aureus were able to recognize two quinolone-resistant and one quinolone-sensitive S. aureus strains showing a perfect correlation with the susceptibility to quinolones.
The complete assay for the specific detection of S. aureus and its susceptibility to quinolone contains the Staphylococcus-specific primers (SEQ ID NOs. 553 and 575) described in Example 7 and the multiplex containing the S. aureus gyrA- and parC-specific primer pairs (SEQ ID NOs. 1340-1341 and SEQ ID NOs. 1342-1343). Amplification is coupled with post-PCR hybridization with the internal probe specific to S. aureus (SEQ ID NO. 587) described in Example 7 and the internal probes specific to wild-type S. aureus gyrA and parC (SEQ ID NOs. 1940-1941) and to the S. aureus gyrA and parC variants (SEQ ID NOs. 1333-1338).
An assay was also developed for the detection of quinolone-resistant S. aureus using the SmartCycler (Cepheid). Real-time detection is based on the use of S. aureus parC-specific primers (SEQ ID NOs. 1342 and 1343) and the Staphylococcus-specific primers (SEQ ID NOs. 553 and 575) described in Example 7. Internal probes were designed for molecular beacon detection of the wild-type S. aureus parC (SEQ ID NO. 1939), for detection of the Ser-80 to Tyr or Phe amino acid substitutions in the ParC subunit encoded by S. aureus parC (SEQ ID NOs. 1938 and 1955) and for detection of S. aureus (SEQ ID NO. 2282).
Example 34
Development of a PCR Assay for the Detection and Identification of Klebsiella pneumoniae and its Quinolone Resistance Genes gyrA and parC.
The analysis of gyrA and parC sequences from a variety of bacterial species from the public databases and from the database described in Example 32 revealed conserved regions allowing the design of PCR primers specific to the quinolone-resistance-determining region (QRDR) of gyrA and parC from K. pneumoniae. PCR primer pair SEQ ID NOs. 1936 and 1937, or pair SEQ ID NOs. 1937 and 1942, were designed to amplify the gyrA sequence of K. pneumoniae, whereas PCR primer pair SEQ ID NOs. 1934 and 1935 was designed to amplify K. pneumoniae parC sequence. An alternative pair, SEQ ID NOs. 1935 and 1936, can also amplify K. pneumoniae parC. The comparison of gyrA and parC sequences from K. pneumoniae strains with various levels of quinolone resistance allowed the identification of amino acid substitutions Ser-83 to Tyr or Phe and Asp-87 to Gly or Ala and Asp-87 to Asn in the GyrA subunit of DNA gyrase encoded by gyrA and amino acid changes Ser-80 to Ile or Arg and Glu-84 to Gly or Lys in the ParC subunit of topoisomerase IV encoded by parC. These amino acid substitutions in the GyrA and ParC subunits occur in isolates with intermediate- or high-level quinolone resistance. Internal probes for the specific detection of wild-type K. pneumoniae gyrA (SEQ ID NO. 1943) and wild-type K. pneumoniae parC (SEQ ID NO. 1944) as well as internal probes for the specific detection of each of the gyrA (SEQ ID NOs. 1945-1949) and parC mutations identified in quinolone-resistant K. pneumoniae (SEQ ID NOs. 1950-1953) were designed.
Two multiplex using the K. pneumoniae gyrA- and parC-specific primer pairs were used: the first multiplex contained K. pneumoniae gyrA-specific primers (SEQ ID NOs. 1937 and 1942) and K. pneumoniae parC-specific primers (SEQ ID NOs. 1934 and 1935) and the second multiplex contained K. pneumoniae gyrA/parC-specific primer (SEQ ID NOs. 1936), K. pneumoniae gyrA-specific primer (SEQ ID NO. 1937) and K. pneumoniae parC-specific primer (SEQ ID NO. 1935). Standard PCR was carried out on PTC-200 thermocyclers (MJ Research) using for the first multiplex 0.6, 0.6, 0.4, 0.4 μM of each primer, respectively, and for the second multiplex 0.8, 0.4, 0.4 μM of each primer, respectively. PCR amplification and agarose gel electrophoresis of the amplified products were performed as described in Example 28. The optimal cycling conditions for maximum sensitivity and specificity were 3 minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 62° C., followed by terminal extension at 72° C. for 2 minutes. The specificity of the two multiplex assays with 40-cycle PCR was verified by using 0.1 ng of purified genomic DNA from a panel of gram-negative bacteria. The list included: Acinetobacter baumannii, Citrobacter freundii, Eikenella corrodens, Enterobacter aerogenes, Enterobacter cancerogenes, Enterobacter cloacae, Escherichia coli (10 strains), Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ornitholytica, Klebsiella oxytoca (2 strains), Klebsiella planticola, Klebsiella terrigena, Kluyvera ascorbata, Kluyvera cryocrescens, Kluyvera georgiana, Neisseria gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella choleraesuis subsp. typhimurium, Salmonella enteritidis, Serratia liquefaciens, Serratia marcescens and Yersinia enterocolytica. For both multiplex, strong amplification of both gyrA and parC was observed only for the K. pneumoniae strain tested. The sensitivity of the two multiplex assays with 40-cycle PCR was verified with one quinolone-sensitive strain of K. pneumoniae. The detection limit was around 10 copies of genomic DNA.
The complete assay for the specific detection of K. pneumoniae and its susceptibility to quinolone contains the Klebsiella-specific primers (SEQ ID NOs. 1331 and 1332) described in Example 29 and either the multiplex containing the K. pneumoniae gyrA- and parC-specific primers (SEQ ID NOs. 1935, 1936, 1937) or the multiplex containing the K. pneumoniae gyrA- and parC-specific primers (SEQ ID NOs. 1934, 1937, 1939, 1942). Amplification is coupled with post-PCR hybridization with the internal probe specific to K. pneumoniae (SEQ ID NO. 2167) described in Example 29 and the internal probes specific to wild-type K. pneumoniae gyrA and parC (SEQ ID NOs. 1943, 1944) and to the K. pneumoniae gyrA and parC variants (SEQ ID NOs. 1945-1949 and 1950-1953).
An assay was also developed for the detection of quinolone-resistant K. pneumoniae using the SmartCycler (Cepheid). Real-time detection is based on the use of resistant K. pneumoniae gyrA-specific primers (SEQ ID NOs. 1936 and 1937) and the K. pneumoniae-specific primers (SEQ ID NOs. 1331 and 1332) described in Example 29. Internal probes were designed for molecular beacon detection of the wild-type K. pneumoniae gyrA (SEQ ID NO. 2251), for detection of the Ser-83 to Tyr or Phe and/or Asp-87 to Gly or Asn in the GyrA subunit of DNA gyrase encoded by gyrA (SEQ ID NOs. 2250) and for detection of K. pneumoniae (SEQ ID NO. 2281).
Example 35
Development of a PCR Assay for Detection and Identification of S. pneumoniae and its Quinolone Resistance Genes gyrA and parC.
The analysis of gyrA and parC sequences from a variety of bacterial species revealed conserved regions allowing the design of PCR primers able to amplify the quinolone-resistance-determining region (QRDR) of gyrA and parC from all S. pneumoniae strains. PCR primer pair SEQ ID NOs. 2040 and 2041 was designed to amplify the QRDR of S. pneumoniae gyrA, whereas PCR primer pair SEQ ID NOs. 2044 and 2045 was designed to amplify the QRDR of S. pneumoniae parC. The comparison of gyrA and parC sequences from S. pneumoniae strains with various levels of quinolone resistance allowed the identification of amino acid substitutions Ser-81 to Phe or Tyr in the GyrA subunit of DNA gyrase encoded by gyrA and amino acid changes Ser-79 to Phe in the ParC subunit of topoisomerase IV encoded by parC. These amino acid substitutions in the GyrA and ParC subunits occur in isolates with intermediate- or high-level quinolone resistance. Internal probes for the specific detection of each of the gyrA (SEQ ID NOs. 2042 and 2043) and parC (SEQ ID NO. 2046) mutations identified in quinolone-resistant S. pneumoniae were designed.
For all bacterial species, amplification was performed from purified genomic DNA. 1 μl of genomic DNA at 0.1 ng/μL was transferred directly to a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.4 μM (each) of the above primers SEQ ID NOs. 2040, 2041, 2044 and 2045, 0.05 mM bovine serum albumin (BSA) and 0.5 U Taq polymerase coupled with TaqStartTM antibody. The optimal cycling conditions for maximum sensitivity and specificity were 3 minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 58° C., followed by terminal extension at 72° C. for 2 minutes. In order to generate Digoxigenin (DIG)-labeled amplicons for capture probe hybridization, 0.1×PCR DIG labeling four deoxynucleoside triphosphates mix (Boehringer Mannheim GmbH) was used for amplification.
The DIG-labeled amplicons were hybridized to the capture probes bound to 96-well plates. The plates were incubated with anti-DIG-alkaline phosphatase and the chemiluminescence was measured by using a luminometer (MLX, Dynex Technologies Inc.) after incubation with CSPD and recorded as Relative Light Unit (RLU). The RLU ratio of tested sample with and without captures probes was then calculated. A ratio ≧2.0 was defined as a positive hybridization signal. All reactions were performed in duplicate.
The specificity of the multiplex assay with 40-cycle PCR was verified by using 0.1 ng of purified genomic DNA from a panel of bacteria listed in Table 13. Strong amplification of both gyrA and parC was detected only for the S. pneumoniae strains tested. Weak amplification of both gyrA and parC genes was detected for Staphylococcus simulans. The detection limit tested with purified genomic DNA from 5 strains of S. pneumoniae was 1 to 10 genome copies. In addition, 5 quinolone-resistant and 2 quinolone-sensitive clinical isolates of S. pneumoniae were tested to further validate the developed multiplex PCR coupled with capture probe hybridization assays. There was a perfect correlation between detection of S. pneumoniae gyrA and parC mutations and the susceptibility to quinolone.
The complete assay for the specific detection of S. pneumoniae and its susceptibility to quinolone contains the S. pneumoniae-specific primers (SEQ ID NOs. 1179 and 1181) described in Example 20 and the multiplex containing the S. pneumoniae gyrA-specific and parC-specific primer pairs (SEQ ID NOS. 2040 and 2041 and SEQ ID NOs. 2044 and 2045). Amplification is coupled with post-PCR hybridization with the internal probe specific to S. pneumoniae (SEQ ID NO. 1180) described in Example and the internal probes specific to each of the S. pneumoniae gyrA and parC variants (SEQ ID NOs. 2042, 2043 and 2046).
Example 36
Detection of Extended-Spectrum TEM-Type β-Lactamases in Escherichia coli. 
The analysis of TEM sequences which confer resistance to third-generation cephalosporins and to β-lactamase inhibitors allowed the identification of amino acid substitutions Met-69 to Ile or Leu or Val, Ser-130 to Gly, Arg-164 to Ser or His, Gly-238 to Ser, Glu-240 to Lys and Arg-244 to Ser or Cys or Thr or His or Leu. PCR primers SEQ ID NOs. 1907 and 1908 were designed to amplify TEM sequences. Internal probes for the specific detection of wild-type TEM (SEQ ID NO. 2141) and for each of the amino acid substitutions (SEQ ID NOs. 1909-1926) identified in TEM variants were designed to detect resistance to third-generation cephalosporins and to β-lactamase inhibitors. Design and synthesis of primers and probes, and detection of the hybridization were performed as described in Example 7.
For all bacterial species, amplification was performed from purified genomic DNA. One μl of genomic DNA at 0.1 ng/μl was transferred directly to a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0); 0.1% Triton X-100, 2.5 mM MgCl2, 0.4 μM of the TEM-specific primers SEQ ID NOs. 1907 and 1908, 200 μM (each) of the four deoxynucleoside triphosphates, 0.05 mM bovine serum albumin (BSA) and 0.5 U Taq polymerase (Promega) coupled with TaqStartTM antibody. PCR amplification and agarose gel analysis of the amplified products were performed as described in Example 28. The optimal cycling conditions for maximum sensitivity and specificity were 3 minutes at 95° C. for initial denaturation, then forty cycles of three steps consisting of 5 seconds at 95° C., 30 seconds at 55° C. and 30 seconds at 72° C., followed by terminal extension at 72° C. for 2 minutes.
The specificity of the TEM-specific primers with 40-cycle PCR was verified by using 0.1 ng of purified genomic from the following bacteria: three third-generation cephalosporin-resistant Escherichia coli strains (one with TEM-10, one with TEM-28 and the other with TEM-49), two third-generation cephalosporin-sensitive Escherichia coli strain (one with TEM-1 and the other without TEM), one third-generation cephalosporin-resistant Klebsiella pneumoniae strain (with TEM-47), and one β-lactamase-inhibitor-resistant Proteus mirabilis strain (with TEM-39). Amplification with the TEM-specific primers was detected only for strains containing TEM.
The sensitivity of the assay with 40-cycle PCR was verified with three E. coli strains containing TEM-1 or TEM-10 or TEM-49, one K. pneumoniae strain containing TEM-47 and one P. mirabilis strain containing TEM-39. The detection limit was 5 to 100 copies of genomic DNA, depending on the TEM-containing strains tested.
The TEM-specific primers SEQ ID NOs. 1907 and 1908 were used in multiplex with the Escherichia coli/Shigella sp.-specific primers SEQ ID NOs. 1661 and 1665 described in Example 28 to allow the complete identification of Escherichia coli/Shigella sp. and the susceptibility to β-lactams. PCR amplification with 0.4 μM of each of the primers and agarose gel analysis of the amplified products was performed as described above.
The specificity of the multiplex with 40-cycle PCR was verified by using 0.1 ng of purified genomic DNA from the following bacteria: three third-generation cephalosporin-resistant Escherichia coli strains (one with TEM-10, one with TEM-28 and the other with TEM-49), two third-generation cephalosporin-sensitive Escherichia coli strain (one with TEM-1 and the other without TEM), one third-generation cephalosporin-resistant Klebsiella pneumoniae strain (with TEM-47), and one β-lactamase-inhibitor-resistant Proteus mirabilis strain (with TEM-39). The multiplex was highly specific to Escherichia coli strains containing TEM.
The complete assay for detection of TEM-type β-lactamases in E. coli includes PCR amplification using the multiplex containing the TEM-specific primers (SEQ ID NOs. 1907 and 1908) and the Escherichia coli/Shigella sp.-specific primers (SEQ ID NOs. 1661 and 1665) coupled with post PCR-hybridization with the internal probes specific to wild-type TEM (SEQ ID NO. 2141) and to the TEM variants (SEQ ID NOs. 1909-1926).
Example 37
Detection of Extended-spectrum SHV-type β-Lactamases in Klebsiella pneumoniae. 
The comparison of SHV sequences, which confer resistance to third-generation cephalosporins and to β-lactamase inhibitors, allowed the identification of amino acid substitutions Ser-130 to Gly, Asp-179 to Ala or Asn, Gly-238 to Ser, and Glu-240 to Lys. PCR primer pair SEQ ID NOs. 1884 and 1885 was designed to amplify SHV sequences. Internal probes for the specific identification of wild-type SHV (SEQ ID NO. 1896) and for each of the amino acid substitutions (SEQ ID NOs. 1886-1895 and 1897-1898) identified in SHV variants were designed to detect resistance to third-generation cephalosporins and to β-lactamase inhibitors. Design and synthesis of primers and probes, and detection of the hybridization were performed as described in Example 7.
For all bacterial species, amplification was performed from purified genomic DNA. One μl of genomic DNA at 0.1 ng/μl was transferred directly to a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.4 μM of the SHV-specific primers SEQ ID NO. 1884 and 1885, 200 μM (each) of the four deoxynucleoside triphosphates, 0.05 mM bovine serum albumin (BSA) and 0.5 U Taq polymerase (Promega) coupled with TaqStartTM antibody. PCR amplification and agarose gel analysis of the amplified products were performed as described in Example 28. The optimal cycling conditions for maximum sensitivity and specificity were 3 minutes at 95° C. for initial denaturation, then forty cycles of three steps consisting of 5 seconds at 95° C., 30 seconds at 55° C. and 30 seconds at 72° C., followed by terminal extension at 72° C. for 2 minutes.
The specificity of the SHV-specific primers with 40-cycle PCR was verified by using 0.1 ng of purified genomic from the following bacteria: two third-generation cephalosporin-resistant Klebsiella pneumoniae strains (one with SHV-2a and the other with SHV-12), one third-generation cephalosporin-sensitive Klebsiella pneumoniae strain (with SHV-1), two third-generation cephalosporin-resistant Escherichia coli strains (one with SHV-8 and the other with SHV-7), and two third-generation cephalosporin-sensitive Escherichia coli strains (one with SHV-1 and the other without any SHV). Amplification with the SHV-specific primers was detected only for strains containing SHV.
The sensitivity of the assay with 40-cycle PCR was verified with four strains containing SHV. The detection limit was 10 to 100 copies of genomic DNA, depending on the SHV-containing strains tested.
The amplification was coupled with post-PCR hybridization with the internal probes specific for identification of wild-type SHV (SEQ ID NO. 1896) and for each of the amino acid substitutions (SEQ ID NOs. 1886-1895 and 1897-1898) identified in SHV variants. The specificity of the probes was verified with six strains containing various SHV enzymes, one Klebsiella pneumoniae strain containing SHV-1, one Klebsiella pneumoniae strain containing SHV-2a, one Klebsiella pneumoniae strain containing SHV-12, one Escherichia coli strain containing SHV-1, one Escherichia coli strain containing SHV-7 and one Escherichia coli strain containing SHV-8. The probes correctly detected each of the SHV genes and their specific mutations. There was a perfect correlation between the SHV genotype of the strains and the susceptibility to β-lactam antibiotics.
The SHV-specific primers SEQ ID NOs. 1884 and 1885 were used in multiplex with the K. pneumoniae-specific primers SEQ ID NOs. 1331 and 1332 described in Example 29 to allow the complete identification of K. pneumoniae and the susceptibility to β-lactams. PCR amplification with 0.4 μM of each of the primers and agarose gel analysis of the amplified products were performed as described above.
The specificity of the multiplex with 40-cycle PCR was verified by using 0.1 ng of purified genomic DNA from the following bacteria: three K. pneumoniae strains containing SHV-1, one Klebsiella pneumoniae strain containing SHV-2a, one Klebsiella pneumoniae strain containing SHV-12, one K. rhinoscleromatis strain containing SHV-1, one Escherichia coli strain without SHV. The multiplex was highly specific to Klebsiella pneumoniae strain containing SHV.
Example 38
Development of a PCR Assay for the Detection and Identification of Neisseria gonorrhoeae and its Associated Tetracycline Resistance Gene tetM.
The analysis of publicly available tetM sequences revealed conserved regions allowing the design of PCR primers specific to tetM sequences. The PCR primer pair SEQ ID NOs. 1588 and 1589 was used in multiplex with the Neisseria gonorrhoeae-specific primers SEQ ID NOs. 551 and 552 described in Example 31. Sequence alignment analysis of tetM sequences revealed regions suitable for the design of an internal probe specific to tetM (SEQ ID NO. 2254). PCR amplification was carried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM of each primer pair as described in Example 28. The optimal cycling conditions for maximum sensitivity and specificity were as follow: three minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 60° C., followed by terminal extension at 72° C. for 2 minutes.
The specificity of the multiplex PCR assay with 40-cycle PCR was verified by using 0.1 ng of purified genomic DNA from the following bacteria: two tetracycline-resistant Escherichia coli strains (one containing the tetracycline-resistant gene tetB and the other containing the tetracycline-resistant gene tetC), one tetracycline-resistant Pseudomonas aeruginosa strain (containing the tetracycline-resistant gene tetA), nine tetracycline-resistant Neisseria gonorrhoeae strains, two tetracycline-sensitive Neisseria meningitidis strains, one tetracycline-sensitive Neisseria polysaccharea strain, one tetracycline-sensitive Neisseria sicca strain and one tetracycline-sensitive Neisseria subflava strain. Amplification with both the tetM-specific and Neisseria gonorrhoeae-specific primers was detected only for N. gonorrhoeae strains containing tetM. There was a weak amplification signal using Neisseria gonorrhoeae-specific primers for the following species: Neisseria sicca, Neisseria polysaccharea and Neisseria meningitidis. There was a perfect correlation between the tetM genotype and the tetracycline susceptibility pattern of the Neisseria gonorrhoeae strains tested. The internal probe specific to N. gonorrhoeae SEQ ID NO. 2166 described in Example 31 can discriminate Neisseria gonorrhoeae from the other Neisseria sp.
The sensitivity of the assay with 40-cycle PCR was verified with two tetracycline resistant strains of N. gonorrhoeae. The detection limit was 5 copies of genomic DNA for both strains.
Example 39
Development of a PCR Assay for the Detection and Identification of Shigella sp. and their Associated Trimethoprim Resistance Gene dhfrIa.
The analysis of publicly available dhfrIa and other dhfr sequences revealed regions allowing the design of PCR primers specific to dhfrIa sequences. The PCR primer pair (SEQ ID NOs. 1459 and 1460) was used in multiplex with the Escherichia coli/Shigella sp.-specific primers SEQ ID NOs. 1661 and 1665 described in Example 28. Sequence alignment analysis of dhfrIa sequences revealed regions suitable for the design of an internal probe specific to dhfrIa (SEQ ID NO. 2253). PCR amplification and agarose gel analysis of the amplified products were performed as described in Example 28 with an annealing temperature of 60° C. The specificity of the multiplex assay with 40-cycle PCR was verified by using 0.1 ng of purified genomic DNA from a panel of bacteria. The list included the following trimethoprim-sensitive strains, Salmonella typhimyurium, Salmonella typhi, Salmonella enteritidis, Tatumella ptyseos, Klebsiella pneumoniae, Enterobacter aerogenes, Citrobacter farmeri, Campylobacter jejuni, Serratia marcescens, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, six trimethoprim-resistant Escherichia coli strains (containing dhfrIa or dhfrV or dhfrVII or dhfrXII or dhfrXIII or dhfrXV), four trimethoprim-resistant strains containing dhfrIa (Shigella sonnei, Shigella flexneri, Shigella dysenteriae and Escherichia coli). There was a perfect correlation between the dhfrIa genotype and the trimethoprim susceptibility pattern of the Escherichia coli and Shigella sp. strains tested. The dhfrIa primers were specific to the dhfrIa gene and did not amplify any of the other trimethoprim-resistant dhfr genes tested. The sensitivity of the multiplex assay with 40-cycle PCR was verified with three strains of trimethoprim-resistant strains of Shigella sp. The detection limit was 5 to 10 genome copies of DNA, depending on the Shigella sp. strains tested.
Example 40
Development of a PCR Assay for the Detection and Identification of Acinetobacter baumannii and its Associated Aminoglycoside Resistance Gene aph(3′)-Via.
The comparison of publicly available aph(3′)-VIa sequence revealed regions allowing the design of PCR primers specific to aph(3′)-VIa. The PCR primer pair (SEQ ID NOs. 1404 and 1405) was used in multiplex with the Acinetobacter baumannii-specific primers SEQ ID NOs. 1692 and 1693 described in Example 30. Analysis of the aph(3′)-VIa sequence revealed region suitable for the design of an internal probe specific to aph(3′)-VIa (SEQ ID NO. 2252). PCR amplification and agarose gel analysis of the amplified products were performed as described in Example 28. The specificity of the multiplex assay with 40-cycle PCR was verified by using 0.1 ng of purified genomic DNA from a panel of bacteria including: two aminoglycoside-resistant A. baumannii strains (containing aph(3′)-VIa), one aminoglycoside-sensitive A. baumanii strain, one of each of the following aminoglycoside-resistant bacteria, one Serratia marcescens strain containing the aminoglycoside-resistant gene aacC1, one Serratia marcescens strain containing the aminoglycoside-resistant gene aacC4, one Enterobacter cloacae strain containing the aminoglycoside-resistant gene aacC2 one Enterococcus faecalis containing the aminoglycoside-resistant gene aacA-aphD, one Pseudomonas aeruginosa strain containing the aminoglycoside-resistant gene aac6IIa and one of each of the following aminoglycoside-sensitive bacterial species, Acinetobacter anitratus, Acinetobacter lwoffi, Psychobbacter phenylpyruvian, Neisseria gonorrhoeae, Haemophilus haemolyticus, Haemophilus influenzae, Yersinia enterolitica, Proteus vulgaris, Eikenella corrodens, Escherichia coli. There was a perfect correlation between the aph(3′)-VIa genotype and the aminoglycoside susceptibility pattern of the A. baumannii strains tested. The aph(3′)-VIa-specific primers were specific to the aph(3′)-VIa gene and did not amplify any of the other aminoglycoside-resistant genes tested. The sensitivity of the multiplex assay with 40-cycle PCR was verified with two strains of aminoglycoside-resistant strains of A. baumannii. The detection limit was 5 genome copies of DNA for both A. baumannii strains tested.
Example 41
Specific Identification of Bacteroides fragilis Using atpD (V-type) Sequences.
The comparison of atpD (V-type) sequences from a variety of bacterial species allowed the selection of PCR primers for Bacteroides fragilis. The strategy used to design the PCR primers was based on the analysis of a multiple sequence alignment of various atpD sequences from B. fragilis, as well as atpD sequences from the related species B. dispar, bacterial genera and archaea, especially representatives with phylogenetically related atpD sequences. A careful analysis of this alignment allowed the selection of oligonucleotide sequences which are conserved within the target species but which discriminate sequences from other species, especially from closely related species B. dispar, thereby permitting the species-specific and ubiquitous detection and identification of the target bacterial species.
The chosen primer pair, SEQ ID NOs. 2134-2135, produces an amplification product of 231 bp. Standard PCR was carried out on PTC-200 thermocyclers (MJ Research Inc.) using 0.4 μM of each primers pair as described in Example 28. The optimal cycling conditions for maximum sensitivity and specificity were as follows: three minutes at 95° C. for initial denaturation, then forty cycles of two steps consisting of 1 second at 95° C. and 30 seconds at 60° C., followed by terminal extension at 72° C. for 2 minutes.
The format of this assay is not limited to the one described above. A person skilled in the art could adapt the assay for different formats such as PCR with real-time detection using molecular beacon probes. Molecular beacon probes designed to be used in this assay include, but are not limited to, SEQ ID NO. 2136 for the detection of the B. fragilis amplicon.
Example 42
Evidence for Horizontal Gene Transfer in the Evolution of the Elongation Factor Tu in Enterococci.
Overview
The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present depending on the bacterial species. Most low G+C gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, E. casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, and E. raffinosus. For the other six enterococcal species (E. cecorum, E. columbae, E. faecalis, E. sulfureus, E. saccharolyticus, and E. solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all share a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and L. lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.
Introduction
The elongation factor Tu (EF-Tu) is a GTP binding protein playing a central role in protein synthesis. It mediates the recognition and transport of aminoacyl-tRNAs and their positioning to the A-site of the ribosome. The highly conserved function and ubiquitous distribution render the elongation factor a valuable phylogenetic marker among eubacteria and even throughout the archaebacterial and eukaryotic kingdoms. The tuf genes encoding elongation factor Tu are present in various copy numbers per bacterial genome. Most gram-negative bacteria contain two tuf genes. As found in Escherichia coli, the two genes, while being almost identical in sequence, are located in different parts of the bacterial chromosome. However, recently completed microbial genomes revealed that only one tuf gene is found in Helicobacter pylori as well as in some obligate parasitic bacteria, such as Borrelia burgdorferi, Rickettsia prowazekii, and Treponema pallidum, and in some cyanobacteria. In most gram-positive bacteria studied so far, only one tuf gene was found. However, Southern hybridization showed that there are two tuf genes in some clostridia as well as in Streptomyces coelicolor and S. lividans. Up to three tuf-like genes have been identified in S. ramocissimus. 
Although massive prokaryotic gene transfer is suggested to be one of the factors responsible for the evolution of bacterial genomes, the genes encoding components of the translation machinery are thought to be highly conserved and difficult to be transferred horizontally due to the complexity of their interactions. However, a few recent studies demonstrated evidence that horizontal gene transfer has also occurred in the evolution of some genes coding for the translation apparatus, namely, 16S rRNA and some aminoacyl-tRNA synthetases. No further data suggest that such a mechanism is involved in the evolution of the elongation factors. Previous studies concluded that the two copies of tuf genes in the genomes of some bacteria resulted from an ancient event of gene duplication. Moreover, a study of the tuf gene in R. prowazekii suggested that intrachromosomal recombination has taken place in the evolution of the genome of this organism.
To date, little is known about the tuf genes of enterococcal species. In this study, we analyzed partial sequences of tuf genes in 17 enterococcal species, namely, E. avium, E. casseliflavus, E. cecorum, E. columbae, E. dispar, E. durans, E. faecalis, E. faecium, E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, E. raffinosus, E. saccharolyticus, E. solitarius, and E. sulfureus. We report here the presence of two divergent copies of tuf genes in 11 of these enterococcal species. The 6 other species carried a single tuf gene. The evolutionary implications are discussed.
Materials and Methods
Bacterial strains. Seventeen enterococcal strains and other gram-positive bacterial strains obtained from the American Type Culture Collection (ATCC, Manassas, Va.) were used in this study (Table 16). All strains were grown on sheep blood agar or in brain-heart infusion broth prior to DNA isolation.
DNA isolation. Bacterial DNAs were prepared using the G NOME DNA extraction kit (Bio101, Vista, Calif.) as previously described.
Sequencing of putative tuf genes. In order to obtain the tuf gene sequences of enterococci and other gram-positive bacteria, two sequencing approaches were used: 1) sequencing of cloned PCR products and 2) direct sequencing of PCR products. A pair of degenerate primers (SEQ ID NOs. 664 and 697) were used to amplify an 886-bp portion of the tuf genes from enterococcal species and other gram-positive bacteria as previously described. For E. avium, E. casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E. hirae, E. mundtii, E. pseudoavium, and E. raffinosus, the amplicons were cloned using the Original TA cloning kit (Invitrogen, Carlsbad, Calif.) as previously described. Five clones for each species were selected for sequencing. For E. cecorum, E. faecalis, E. saccharolyticus, and E. solitarius as well as the other gram-positive bacteria, the sequences of the 886-bp amplicons were obtained by direct sequencing. Based on the results obtained from the earlier rounds of sequencing, two pairs of primers were designed for obtaining the partial tuf sequences from the other enterococcal species by direct sequencing. One pair of primers (SEQ ID NOs. 543 and 660) were used to amplify the enterococcal tuf gene fragments from E. columbae, E. malodoratus, and E. sulfureus. Another pair of primers (SEQ ID NOs. 664 and 661) were used to amplify the second tuf gene fragments from E. avium, E. malodoratus, and E. pseudoavium. 
Prior to direct sequencing, PCR products were electrophoresed on 1% agarose gel at 120V for 2 hours. The gel was then stained with 0.02% methylene blue for 30 minutes and washed twice with autoclaved distilled water for 15 minutes. The gel slices containing PCR products of the expected sizes were cut out and purified with the QIAquick gel extraction kit (QIAgen Inc., Mississauga, Ontario, Canada) according to the manufacturer's instructions. PCR mixtures for sequencing were prepared as described previously. DNA sequencing was carried out with the Big DyeTM Terminator Ready Reaction cycle sequencing kit using a 377 DNA sequencer (PE Applied Biosystems, Foster City, Calif.). Both strands of the amplified DNA were sequenced. The sequence data were verified using the SequencerTM 3.0 software (Gene Codes Corp., Ann Arbor, Mich.).
Sequence analysis and phylogenetic study. Nucleotide sequences of the tuf genes and their respective flanking regions for E. faecalis, Staphylococcus aureus, and Streptococcus pneumoniae, were retrieved from the TIGR microbial genome database and S. pyogenes from the University of Oklahoma database. DNA sequences and deduced protein sequences obtained in this study were compared with those in all publicly available databases using the BLAST and FASTA programs. Unless specified, sequence analysis was conducted with the programs from GCG package (Version 10; Genetics Computer Group, Madison, Wis.). Sequence alignment of the tuf genes from 74 species representing all three kingdoms of life (Tables 16 and 17) were carried out by use of Pileup and corrected upon visual analysis. The N- and C-termini extremities of the sequences were trimmed to yield a common block of 201 amino acids sequences and equivocal residues were removed. Phylogenetic analysis was performed with the aid of PAUP 4.0b4 written by Dr. David L. Swofford (Sinauer Associates, Inc., Publishers, Sunderland, Mass.). The distance matrix and maximum parsimony were used to generate phylogenetic trees and bootstrap resampling procedures were performed using 500 and 100 replications in each analysis, respectively.
Protein structure analysis. The crystal structures of (i) Thermus aquaticus EF-Tu in complex with Phe-tRNAPhe and a GTP analog and (ii) E. coli EF-Tu in complex with GDP served as templates for constructing the equivalent models for enterococcal EF-Tu. Homology modeling of protein structure was performed using the SWISS-MODEL server and inspected using the SWISS-PDB viewer version 3.1.
Southern hybridization. In a previous study, we amplified and cloned an 803-bp PCR product of the tuf gene fragment from E. faecium. Two divergent sequences of the inserts, which we assumed to be tufA and tufB genes, were obtained. The recombinant plasmid carrying either tufA or tufB sequence was used to generate two probes labeled with Digoxigenin (DIG)-11-dUTP by PCR incorporation following the instructions of the manufacturer (Boehringer Mannheim, Laval, Quebec, Canada). Enterococcal genomic DNA samples (1-2 μg) were digested to completion with restriction endonucleases BglII and XbaI as recommended by the supplier (Amersham Pharmacia Biotech, Mississauga, Ontario, Canada). These restriction enzymes were chosen because no restriction sites were observed within the amplified tuf gene fragments of most enterococci. Southern blotting and filter hybridization were performed using positively charged nylon membranes (Boehringer Mannheim) and QuikHyb hybridization solution (Stratagene Cloning Systems, La Jolla, Calif.) according to the manufacturers' instructions with modifications. Twenty μl of each digestion were electrophoresed for 2 h at 120V on a 0.8% agarose gel. The DNA fragments were denatured with 0.5 M NaOH and transferred by Southern blotting onto a positively charged nylon membrane (Boehringer Mannheim). The filters were pre-hybridized for 15 min and then hybridized for 2 h in the QuikHyb solution at 68° C. with either DIG-labeled probe. Posthybridization washings were performed twice with 0.5×SSC, 1% SDS at room temperature for 15 min and twice in the same solution at 60° C. for 15 min. Detection of bound probes was achieved using disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2′-(5′-chloro)tricyclo(3.3.1.13.7)decan)-4-yl)phenyl phosphate (CSPD) (Boehringer Mannheim) as specified by the manufacturer.
GenBank submission. The GenBank accession numbers for partial tuf gene sequences generated in this study are given in Table 16.
Results
Sequencing and nucleotide sequence analysis. In this study, all gram-positive bacteria other than enterococci yielded a single tuf sequence of 886 bp using primers SEQ ID NOs. 664 and 697 (Table 16). Each of four enterococcal species including E. cecorum, E. faecalis, E. saccharolyticus, and E. solitarius also yielded one 886-bp tuf sequence. On the other hand, for E. avium, E. casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E. hirae, E. mundtii, E. pseudoavium, and E. raffinosus, direct sequencing of the 886-bp fragments revealed overlapping peaks according to their sequence chromatograms, suggesting the presence of additional copies of the tuf gene. Therefore, the tuf gene fragments of these 10 species were cloned first and then sequenced. Sequencing data revealed that two different types of tuf sequences (tufA and tufB) are found in eight of these species including E. casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E. hirae, E. mundtii, and E. raffinosus. Five clones from E. avium and E. pseudoavium yielded only a single tuf sequence. These new sequence data allowed the design of new primers specific for the enterococcal tufA or tufB sequences. Primers SEQ ID NOs. 543 and 660 were designed to amplify only enterococcal tufA sequences and a 694-bp fragment was amplified from all 17 enterococcal species. The 694-bp sequences of tufA genes from E. columbae, E. malodoratus, and E. sulfureus were obtained by direct sequencing using these primers. Primers SEQ ID NOs. 664 and 661 were designed for the amplification of 730-bp portion of tufB genes and yielded the expected fragments from 11 enterococcal species, including E. malodoratus and the 10 enterococcal species in which heterogeneous tuf sequences were initially found. The sequences of the tufB fragments for E. avium, E. malodoratus and E. pseudoavium were determined by direct sequencing using the primers SEQ ID NOs. 664 and 661. Overall, tufA gene fragments were obtained from all 17 enterococcal species but tufB gene fragments were obtained with only 11 enterococcal species (Table 16).
The identities between tufA and tufB for each enterococcal species were 68-79% at the nucleotide level and 81 to 89% at the amino acid level. The tufA gene is highly conserved among all enterococcal species with identities varying from 87% to 99% for DNA and 93% to 99% for amino acid sequences, while the identities among tufB genes of enterococci varies from 77% to 92% for DNA and 91% to 99% for amino acid sequences, indicating their different origins and evolution (Table 18). Since E. solitarius has been transferred to the genus Tetragenococcus, which is also a low G+C gram-positive bacterium, our sequence comparison did not include this species as an enterococcus. G+C content of enterococcal tufA sequences ranged from 40.8% to 43.1%, while that of enterococcal tufB sequences varied from 37.8% to 46.3%. Based on amino acid sequence comparison, the enterococcal tufA gene products share higher identities with those of Abiotrophia adiacens, Bacillus subtilis, Listeria monocytogenes, S. aureus, and S. epidermidis. On the other hand, the enterococcal tufB gene products share higher percentages of amino acid identity with the tuf genes of S. pneumoniae, S. pyogenes and Lactococcus lactis (Table 18).
In order to elucidate whether the two enterococcal tuf sequences encode genuine EF-Tu, the deduced amino acid sequences of both genes were aligned with other EF-Tu sequences available in SWISSPROT (Release 38). Sequence alignment demonstrated that both gene products are highly conserved and carry all conserved residues present in this portion of prokaryotic EF-Tu (FIG. 4). Therefore, it appears that both gene products could fulfill the function of EF-Tu. The partial tuf gene sequences encode the portion of EF-Tu from residues 117 to 317, numbered as in E. coli. This portion makes up of the last four α-helices and two β-strands of domain I, the entire domain II and the N-terminal part of domain III on the basis of the determined structures of E. coli EF-Tu.
Based on the deduced amino acid sequences, the enterococcal tufB genes have unique conserved residues Lys129, Leu140, Ser230, and Asp234 (E. coli numbering) that are also conserved in streptococci and L. lactis, but not in the other bacteria (FIG. 4). All these residues are located in loops except for Ser230. In other bacteria the residue Ser230 is substituted for highly conserved Thr, which is the 5th residue of the third N-strand of domain II. This region is partially responsible for the interaction between the EF-Tu and aminoacyl-tRNA by the formation of a deep pocket for any of the 20 naturally occurring amino acids. According to our three-dimensional model (data not illustrated), the substitution Thr230→Ser in domain II of EF-Tu may have little impact on the capability of the pocket to accommodate any amino acid. However, the high conservation of Thr230 comparing to the unique Ser substitution found only in streptococci and 11 enterococci could suggest a subtle functional role for this residue.
The tuf gene sequences obtained for E. faecalis, S. aureus, S. pneumoniae and S. pyogenes were compared with their respective incomplete genome sequence. Contigs with more than 99% identity were identified. Analysis of the E. faecalis genome data revealed that the single E. faecalis tuf gene is located within an str operon where tuf is preceded by fus that encodes the elongation factor G. This str operon is present in S. aureus and B. subtilis but not in the two streptococcal genomes examined. The 700-bp or so sequence upstream the S. pneumoniae tuf gene has no homology with any known gene sequences. In S. pyogenes, the gene upstream of tuf is similar to a cell division gene, ftsW, suggesting that the tuf genes in streptococci are not arranged in a str operon.
Phylogenetic analysis. Phylogenetic analysis of the tuf amino acid sequences with representatives of eubacteria, archeabacteria, and eukaryotes using neighbor-joining and maximum parsimony methods showed three major clusters representing the three kingdoms of life. Both methods gave similar topologies consistent with the rRNA gene data (data not shown). Within the bacterial clade, the tree is polyphyletic but tufA genes from all enterococcal species always clustered with those from other low G+C gram-positive bacteria (except for streptococci and lactococci), while the tufB genes of the 11 enterococcal species form a distinct cluster with streptococci and L. lactis (FIG. 5). Duplicated genes from the same organism do not cluster together, thereby not suggesting evolution by recent gene duplication.
Southern hybridization. Southern hybridization of BglII/XbaI digested genomic DNA from 12 enterococcal species tested with the tufA probe (DIG-labeled tufA fragment from E. faecium) yielded two bands of different sizes in 9 species, which also carried two divergent tuf sequences according to their sequencing data. For E. faecalis and E. solitarius, a single band was observed indicating that one tuf gene is present (FIG. 6). A single band was also found when digested genomic DNA from S. aureus, S. pneumoniae, and S. pyogenes were hybridized with the tufA probe (data not shown). For E. faecium, the presence of three bands can be explained by the existence of a XbaI restriction site in the middle of the tufA sequence, which was confirmed by sequencing data. Hybridization with the tufB probe (DIG-labeled tufB fragment of E. faecium) showed a banding profile similar to the one obtained with the tufA probe (data not shown).
Discussion
In this study, we have shown that two divergent copies of genes encoding the elongation factor Tu are present in some enterococcal species. Sequence data revealed that both genes are highly conserved at the amino acid level. One copy (tufA) is present in all enterococcal species, while the other (tufB) is present only in 11 of the 17 enterococcal species studied. Based on 16S rRNA sequence analysis, these 11 species are members of three different enterococcal subgroups (E. avium, E. faecium, and E. gallinarum species groups) and a distinct species (E. dispar). Moreover, 16S rDNA phylogeny suggests that these 11 species possessing 2 tuf genes all share a common ancestor before they further evolved to become the modern species. Since the six other species having only one copy diverged from the enterococcal lineage before that common ancestor, it appears that the presence of one tuf gene in these six species is not attributable to gene loss.
Two clusters of low G+C gram-positive bacteria were observed in the phylogenetic tree of the tuf genes: one contains a majority of low G+C gram-positive bacteria and the other contains lactococci and streptococci. This is similar to the finding on the basis of phylogenetic analysis of the 16S rRNA gene and the hrcA gene coding for a unique heat-shock regulatory protein. The enterococcal tufA genes branched with most of the low G+C gram-positive bacteria, suggesting that they originated from a common ancestor. On the other hand, the enterococcal tufB genes branched with the genera Streptococcus and Lactococcus that form a distinct lineage separated from other low G+C gram-positive bacteria (FIG. 5). The finding that these EF-Tu proteins share some conserved amino acid residues unique to this branch also supports the idea that they may share a common ancestor. Although these conserved residues might result from convergent evolution upon a specialized function, such convergence at the sequence level, even for a few residues, seems to be rare, making it an unlikely event. Moreover, no currently known selective pressure, if any, would account for keeping one versus two tuf genes in bacteria. The G+C contents of enterococcal tufA and tufB sequences are similar, indicating that they both originated from low G+C gram-positive bacteria, in accordance with the phylogenetic analysis.
The tuf genes are present in various copy numbers in different bacteria. Furthermore, the two tuf genes are normally associated with characteristic flanking genes. The two tuf gene copies commonly encountered within gram-negative bacteria are part of the bacterial str operon and tRNA-tufB operon, respectively. The arrangement of tufA in the str operon was also found in a variety of bacteria, including Thermotoga maritima, the most ancient bacteria sequenced so far, Aquifex aeolicus, cyanobacteria, Bacillus sp., Micrococcus luteus, Mycobacterium tuberculosis, and Streptomyces sp. Furthermore, the tRNA-tufB operon has also been identified in Aquifex aeolicus, Thermus thermophilus, and Chlamydia trachomatis. The two widespread tuf gene arrangements argue in favor of their ancient origins. It is noteworthy that most obligate intracellular parasites, such as Mycoplasma sp., R. prowazekii, B. burgdorferi, and T. pallidum, contain only one tuf gene. Their flanking sequences are distinct from the two conserved patterns as a result of selection for effective propagation by an extensive reduction in genome size by intragenomic recombination and rearrangement.
Most gram-positive bacteria with low G+C content sequenced to date contain only a single copy of the tuf gene as a part of the str operon. This is the case for B. subtilis, S. aureus and E. faecalis. PCR amplification using a primer targeting a conserved region of the fus gene and the tufA-specific primer SEQ ID NO. 660, but not the tufB-specific primer SEQ ID NO. 661, yielded the expected amplicons for all 17 enterococcal species tested, indicating the presence of the fus-tuf organization in all enterococci (data not shown). However, in the genomes of S. pneumoniae and S. pyogenes, the sequences flanking the tuf genes varies although the tuf gene itself remains highly conserved. The enterococcal tufB genes are clustered with streptococci, but at present we do not have enough data to identify the genes flanking the enterococcal tufB genes. Furthermore, the functional role of the enterococcal tufB genes remains unknown. One can only postulate that the two divergent gene copies are expressed under different conditions.
The amino acid sequence identities between the enterococcal tufA and tufB genes are lower than either i) those between the enterococcal tufA and the tuf genes from other low G+C gram-positive bacteria (streptococci and lactococci excluded) or ii) those between the enterococcal tufB and streptococcal and lactococcal tuf genes. These findings suggest that the enterococcal tufA genes share a common ancestor with other low G+C gram-positive bacteria via the simple scheme of vertical evolution, while the enterococcal tufB genes are more closely related to those of streptococci and lactococci. The facts that some enterococci possess an additional tuf gene and that the single streptococcal tuf gene is not clustered with other low G+C gram-positive bacteria cannot be explained by the mechanism of gene duplication or intrachromosomal recombination. According to sequence and phylogenetic analysis, we propose that the presence of the additional copy of the tuf genes in 11 enterococcal species is due to horizontal gene transfer. The common ancestor of the 11 enterococcal species now carrying tufB genes acquired a tuf gene from an ancestral streptococcus or a streptococcus-related species during enterococcal evolution through gene transfer before the diversification of modern enterococci. Further study of the flanking regions of the gene may provide more clues for the origin and function of this gene in enterococci.
Recent studies of genes and genomes have demonstrated that considerable horizontal transfer occurred in the evolution of aminoacyl-tRNA synthetases in all three kingdoms of life. The heterogeneity of 16S rRNA is also attributable to horizontal gene transfer in some bacteria, such as Streptomyces, Thermomonospora chromogena and Mycobacterium celatum. In this study, we provide the first example in support of a likely horizontal transfer of the tuf gene encoding the elongation factor Tu. This may be an exception since stringent functional constraints do not allow for frequent horizontal transfer of the tuf gene as with other genes. However, enterococcal tuf genes should not be the only such exception as we have noticed that the phylogeny of Streptomyces tuf genes is equally or more complex than that of enterococci. For example, the three tuf-like genes in a high G+C gram-positive bacterium, S. ramocissimus, branched with the tuf genes of phylogenetically divergent groups of bacteria (FIG. 5). Another example may be the tuf genes in clostridia, which represent a phylogenetically very broad range of organisms and form a plethora of lines and groups of various complexities and depths. Four species belonging to three. different clusters within the genus Clostridium have been shown by Southern hybridization to carry two copies of the tuf gene. Further sequence data and phylogenetic analysis may help interpreting the evolution of the elongation factor Tu in these gram-positive bacteria. Since the tuf genes and 16S rRNA genes are often used for phylogenetic study, the existence of duplicate genes originating from horizontal gene transfer may alter the phylogeny of microorganisms when the laterally acquired copy of the gene is used for such analysis. Hence, caution should be taken in interpreting phylogenetic data. In addition, the two tuf genes in enterococci have evolved separately and are distantly related to each other phylogenetically. The enterococcal tufB genes are less conserved and unique to the 11 enterococcal species only. We previously demonstrated that the enterococcal tufA genes could serve as a target to develop a DNA-based assay for identification of enterococci. The enterococcal tufB genes would also be useful in identification of these 11 enterococcal species.
Example 43
Elongation Factor Tu (tuf) and the F-ATPase Beta-Subunit (atpD) as Phylogenetic Tools for Species of the Family Enterobacteriaceae.
Summary
The phylogeny of enterobacterial species commonly found in clinical samples was analyzed by comparing partial sequences of their elongation factor Tu (tuf) genes and their F-ATPase beta-subunit (atpD) genes. A 884-bp fragment for tuf and a 884- or 871-bp fragment for atpD were sequenced for 88 strains of 72 species from 25 enterobacterial genera. The atpD sequence analysis revealed a specific indel to Pantoea and Tatumella species showing for the first time a tight phylogenetic affiliation between these two genera. Comprehensive tuf and atpD phylogenetic trees were constructed and are in agreement with each other. Monophyletic genera are Yersinia, Pantoea, Edwardsiella, Cedecea, Salmonella, Serratia, Proteus, and Providencia. Analogous trees were obtained based on available 16S rDNA sequences from databases. tuf and atpD phylogenies are in agreement with the 16S rDNA analysis despite the smaller resolution power for the latter. In fact, distance comparisons revealed that tuf and atpD genes provide a better resolution for pairs of species belonging to the family Enterobacteriaceae. However, 16S rDNA distances are better resolved for pairs of species belonging to different families. In conclusion, tuf and atpD conserved genes are sufficiently divergent to discriminate different species inside the family Enterobacteriaceae and offer potential for the development of diagnostic tests based on DNA to identify enterobacterial species.
Introduction
Members of the family Enterobacteriaceae are facultatively anaerobic gram-negative rods, catalase-positive and oxydase-positive (Brenner, 1984). They are found in soil, water, plants, and in animals from insects to man. Many enterobacteria are opportunistic pathogens. In fact, members of this family are responsible for about 50% of nosocomial infections in the United States (Brenner, 1984). Therefore, this family is of considerable clinical importance.
Major classification studies on the family Enterobacteriaceae are based on phenotypic traits (Brenner et al., 1999; Brenner et al., 1980; Dickey & Zumoff, 1988; Farmer III et al., 1980; Farmer III et al., 1985b; Farmer III et al., 1985a) such as biochemical reactions and physiological characteristics. However, phenotypically distinct strains may be closely related by genotypic criteria and may belong to the same genospecies (Bercovier et al., 1980; Hartl & Dykhuizen, 1984). Also, phenotypically close strains (biogroups) may belong to different genospecies, like Klebsiella pneumoniae and Enterobacter aerogenes (Brenner, 1984) for example. Consequently, identification and classification of certain species may be ambiguous with techniques based on phenotypic tests (Janda et al., 1999; Kitch et al., 1994; Sharma et al., 1990).
More advances in the classification of members of the family Enterobacteriaceae have come from DNA-DNA hybridization studies (Brenner et al., 1993; Brenner et al., 1986; Brenner, et al., 1980; Farmer III, et al., 1980; Farmer III, et al., 1985b; Izard et al., 1981; Steigerwalt et al., 1976). Furthermore, the phylogenetic significance of bacterial classification based on 16S rDNA sequences has been recognized by many workers (Stackebrandt & Goebel, 1994; Wayne et al., 1987). However, members of the family Enterobacteriaceae have not been subjected to extensive phylogenetic analysis of 16S rDNA (Sproer et al., 1999). In fact, this molecule was not thought to solve taxonomic problems concerning closely related species because of its very high degree of conservation (Brenner, 1992; Sproer, et al., 1999). Another drawback of the 16S rDNA gene is that it is found in several copies within the genome (seven in Escherichia coli and Salmonella typhimurium) (Hill & Harnish, 1981). Due to sequence divergence between the gene copies, direct sequencing of PCR products is often not suitable to achieve a representative sequence (Cilia et al., 1996; Hill & Harnish, 1981). Other genes such as gap and ompA (Lawrence et al., 1991), rpoB (Mollet et al., 1997), and infB (Hedegaard et al., 1999) were used to resolve the phylogeny of enterobacteria. However, none of these studies covered an extensive number of species.
tuf and atpD are the genes encoding the elongation factor Tu (EF-Tu) and the F-ATPase beta-subunit, respectively. EF-Tu is involved in peptide chain formation (Ludwig et al., 1990). The two copies of the tuf gene (tufA and tufB) found in enterobacteria (Sela et al., 1989) share high identity level (99%) in Salmonella typhimurium and in E. coli. The recombination phenomenon could explain sequence homogenization between the two copies (Abdulkarim & Hughes, 1996; Grunberg-Manago, 1996). F-ATPase is present on the plasma membranes of eubacteria (Nelson & Taiz, 1989). It functions mainly in ATP synthesis (Nelson & Taiz, 1989) and the beta-subunit contains the catalytic site of the enzyme. EF-Tu and F-ATPase are highly conserved throughout evolution and shows functional constancy (Amann et al., 1988; Ludwig, et al., 1990). Recently, phylogenies based on protein sequences from EF-Tu and F-ATPase beta-subunit showed good agreement with each other and with the rDNA data (Ludwig et al., 1993).
We elected to sequence 884-bp fragments of tuf and atpD from 88 clinically relevant enterobacterial strains representing 72 species from 25 genera. These sequences were used to create phylogenetic trees that were compared with 16S rDNA trees. These trees revealed good agreement with each others and demonstrated the high resolution of tuf and atpD phylogenies at the species level.
Materials and Methods
Bacterial strains and genomic material. All bacterial strains used in this study were obtained from the American Type Culture Collection (ATCC) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). These enterobacteria can all be recovered from clinical specimens, but not all are pathogens. Whenever possible, we choose type strains. Identification of all strains was confirmed by classical biochemical tests using the automated system MicroScan WalkAway-96 system equipped with a Negative BP Combo Panel Type 15 (Dade Behring Canada). Genomic DNA was purified using the G NOME DNA kit (Bio 101). Genomic DNA from Yersinia pestis was kindly provided by Dr. Robert R. Brubaker. Strains used in this study and their descriptions are shown in Table 19.
PCR primers. The eubacterial tuf and atpD gene sequences available from public databases were analyzed using the GCG package (version 8.0) (Genetics Computer Group). Based on multiple sequence alignments, two highly conserved regions were chosen for each genes, and PCR primers were derived from these regions with the help of Oligo primer analysis software (version 5.0) (National Biosciences). A second 5′ primer was design to amplify the gene atpD for few enterobacteria difficult to amplify with the first primer set. When required, the primers contained inosines or degeneracies to account for variable positions. Oligonucleotide primers were synthesized with a model 394 DNA/RNA synthesizer (PE Applied Biosystems). PCR primers used in this study are listed in Table 20.
DNA sequencing. An 884-bp portion of the tuf gene and an 884-bp portion (or alternatively an 871-bp portion for a few enterobacterial strains) of the atpD gene were sequenced for all enterobacteria listed in the first strain column of Table 19. Amplification was performed with 4 ng of genomic DNA. The 40-μl PCR mixtures used to generate PCR products for sequencing contained 1•0 μM each primer, 200 μM each deoxyribonucleoside triphosphate (Pharmacia Biotech), 10 mM Tris-HCl (pH 9•0 at 25° C.), 50 mM KCl, 0•1% (w/v) Triton X-100, 2•5 mM MgCl2, 0•05 mM BSA, 0•3 U of Taq DNA polymerase (Promega) coupled with TaqStartTM antibody (Clontech Laboratories). The TaqStartTM neutralizing monoclonal antibody for Taq DNA polymerase was added to all PCR mixtures to enhance efficiency of amplification (Kellogg et al., 1994). The PCR mixtures were subjected to thermal cycling (3 min at 95° C. and then 35 cycles of 1 min at 95° C., 1 min at 55° C. for tuf or 50° C. for atpD, and 1 min at 72° C., with a 7-min final extension at 72° C.) using a PTC-200 DNA Engine thermocycler (MJ Research). PCR products having the predicted sizes were recovered from an agarose gel stained for 15 min with 0•02% of methylene blue followed by washing in sterile distilled water for 15 min twice (Flores et al., 1992). Subsequently, PCR products having the predicted sizes were recovered from gels using the QIAquick gel extraction kit (QIAGEN).
Both strands of the purified amplicons were sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) on an automated DNA sequencer (Model 377). Amplicons from two independent PCR amplifications were sequenced for each strain to ensure the absence of sequencing errors attributable to nucleotide miscorporations by the Taq DNA polymerase. Sequence assembly was performed with the aid of Sequencher 3.0 software (Gene Codes).
Phylogenetic analysis. Multiple sequence alignments were performed using PileUp from the GCG package (Version 10.0) (Genetics Computer Group) and checked by eye with the editor SeqLab to edit sequences if necessary and to note which regions were to be excluded for phylogenetic analysis. Vibrio cholerae and Shewanella putrefaciens were used as outgroups. Bootstrap subsets (750 sets) and phylogenetic trees were generated with the Neighbor Joining algorithm from Dr. David Swofford's PAUP (Phylogenetic Analysis Using Parsimony) Software version 4.0b4 (Sinauer Associates) and with tree-bisection branch-swapping. The distance model used was Kimura (1980) two-parameter. Relative rate test was performed with the aid of Phyltest program version 2.0 (c).
Results and Discussion
DNA Amplification, Sequencing and Sequence Alignments
A PCR product of the expected size of 884 bp was obtained for tuf and of 884 or 871 bp for atpD from all bacterial strains tested. After subtracting for biased primer regions and ambiguous single strand data, sequences of at least 721 bp for tuf and 713 bp for atpD were submitted to phylogenetic analyses. These sequences were aligned with tuf and atpD sequences available in databases to verify that the nucleotide sequences indeed encoded a part of tested genes. Gaps were excluded to perform phylogenetic analysis.
Signature Sequences
From the sequence alignments obtained from both tested genes, only one insertion was observed. This five amino acids insertion is located between the positions 325 and 326 of atpD gene of E. coli strain K-12 (Saraste et al., 1981) and can be considered a signature sequence of Tatumella ptyseos and Pantoea species (FIG. 7). The presence of a conserved indel of defined length and sequence and flanked by conserved regions could suggest a common ancestor, particularly when members of a given taxa share this indel (Gupta, 1998). To our knowledge, high relatedness between the genera Tatumella and Pantoea is demonstrated for the first time.
Enterobacter agglomerans ATCC 27989 sequence does not possess the five amino acid indel (FIG. 7). This indel could represent a useful marker to help resolve the Enterobacter agglomerans and Pantoea classification. Indeed, the transfer of Enterobacter agglomerans to Pantoea agglomerans was proposed in 1989 by Gavini et al. (Gavini et al., 1989). However, some strains are provisionally classified as Pantoea sp. until their interrelatedness is elucidated (Gavini, et al., 1989). Since the transfer was proposed, the change of nomenclature has not yet been made for all Enterobacter agglomerans in the ATCC database. The absence of the five amino acids indel suggests that some strains of Enterobacter agglomerans most likely do not belong to the genus Pantoea. 
Phylogenetic Trees Based on Partial Tuf Sequences, Atpd Sequences, and Published 16S Rdna Data of Members of the Enterobacteriaceae.
Representative trees constructed from tuf and atpD sequences with the neighbor-joining method are shown in FIG. 8. The phylogenetic trees generated from partial tuf sequences and atpD sequences are very similar. Nevertheless, atpD tree shows more monophyletic groups corresponding to species that belong to the same genus. These groups are more consistent with the actual taxonomy. For both genes, some genera are not monophyletic. These results support previous phylogenies based on the genes gap and ompA (Lawrence, et al., 1991), rpoB (Mollet, et al., 1997), and infB (Hedegaard, et al., 1999) which all showed that the genera Escherichia and Klebsiella are polyphyletic. There were few differences in branching between tuf and atpD genes.
Even though Pantoea agglomerans and Pantoea dispersa indels were excluded for phylogenetic analysis, these two species grouped together and were distant from Enterobacter agglomerans ATCC 27989, adding another evidence that the latter species is heterogenous and that not all members of this species belong to the genus Pantoea. In fact, the E. agglomerans strain ATCC 27989 exhibits branch lengths similar to others Enterobacter species with both genes. Therefore, we suggest that this strain belong to the genus Enterobacter until further reclassification of that genus.
tuf and atpD trees exhibit very short genetic distances between taxa belonging to the same genetic species including species segregated for clinical considerations. This first concern E. coli and Shigella species that were confirmed to be the same genetic species by hybridization studies (Brenner et al., 1972; Brenner et al., 1972; Brenner et al., 1982) and phylogenies based on 16S rDNA (Wang et al., 1997) and rpoB genes (Mollet, et al., 1997). Hybridization studies (Bercovier, et al., 1980) and phylogeny based on 16S rDNA genes (Ibrahim et al., 1994) demonstrated also that Yersinia pestis and Y. pseudotuberculosis are the same genetic species. Among Yersinia pestis and Y. pseudotuberculosis, the three Klebsiella pneumoniae subspecies, E. coli-Shigella species, and Salmonella choleraesuis subspecies, Salmonella is a less tightly knit species than the other genetic species. The same is true for E. coli and Shigella species.
Escherichia fergusonii is very close to E. coli-Shigella genetic species. This observation is corroborated by 16S rDNA phylogeny (McLaughlin et al., 2000) but not by DNA hybridization values. In fact, E. fergusonii is only 49% to 63% related to E. coli-Shigella (Farmer III, et al., 1985b). It was previously observed that very recently diverged species may not be recognizable based on 16S rDNA sequences although DNA hybridization established them as different species (Fox et al., 1992). Therefore, E. fergusonii could be a new “quasi-species”.
atpD phylogeny revealed Salmonella subspecies divisions consistent with the actual taxonomy. This result was already observed by Christensen et al. (Christensen & Olsen; 1998). Nevertheless, tuf partial sequences discriminate less than atpD between Salmonella subspecies.
Overall, tuf and atpD phylogenies exhibit enough divergence between species to ensure efficient discrimination. Therefore, it could be easy to distinguish phenotypically close enterobacteria belonging to different genetic species such as Klebsiella pneumoniae and Enterobacter aerogenes. 
Phylogenetic relationships between Salmonella, E. coli and C. freundii are not well defined. 16S rDNA and 23S rDNA sequence data reveals a closer relationship between Salmonella and E. coli than between Salmonella and C. freundii (Christensen et al., 1998), while DNA homology studies (Selander et al., 1996) and infb phylogeny (Hedegaard, et al., 1999) showed that Salmonella is more closely related to C. freundii than to E. coli. In that regard, tuf and atpD phylogenies are coherent with 16S rDNA and 23S rDNA sequence analysis.
Phylogenetic analyses were also performed using amino acids sequences. tuf tree based on amino acids is characterized by a better resolution between taxa outgroup and taxa ingroup (enterobacteria) than tree based on nucleic acids whereas atpD trees based on amino acids and nucleic acids give almost the same resolution between taxa outgroup and ingroup (data not shown).
Relative rate test (or two cluster test (Takezaki et al., 1995)) evaluates if evolution is constant between two taxa. Before to apply the test, the topology of a tree is determined by tree-building method without the assumption of rate constancy. Therefore, two taxa (or two groups of taxa) are compared with a third taxon that is an outgroup of the first two taxa (Takezaki, et al., 1995). Few pairs of taxa that exhibited a great difference between their branch lengths at particular nodes were chosen to perform the test. This test reveals that tuf and atpD are not constant in their evolution within the family Enterobacteriaceae. For tuf, for example, the hypothesis of rate constancy is rejected (Z value higher than 1-96) between Yersinia species. The same is true for Proteus species. For atpD, for example, evolution is not constant between Proteus species, between Proteus species and Providencia species, and between Yersinia species and Escherichia coli. For 16S rDNA, for example, evolution is not constant between two E. coli, between E. coli and Enterobacter aerogenes, and between E. coli and Proteus vulgaris. These results suggest that tuf, atpD and 16S rDNA could not serve as a molecular clock for the entire family Enterobacteriaceae.
Since the number and the nature of taxa can influence topology of trees, phylogenetic trees from tuf and atpD were reconstructed using sequences corresponding to strains for which 16S rDNA genes were published in GenEMBL. These trees were similar to those generated using 16S rDNA (FIG. 9). Nevertheless, 16S rDNA tree gave poorer resolution power than tuf and atpD gene trees. Indeed, these latter exhibited less multifurcation (polytomy) than the 16S rDNA tree.
Comparison of Distances Based on tuf, atpD, and 16S rDNA Data
tuf, atpD, and 16S rDNA distances (i.e. the number of differences per nucleotide site) were compared with each other for each pair of strains. We found that the tuf and atpD distances were respectively 2•268±0•965 and 2•927±0•896 times larger than 16S rDNA distances (FIGS. 10a and b). atpD distances were 1•445±0•570 times larger than tuf distances (FIG. 10c). FIG. 10 also shows that the tuf, atpD, and 16S rDNA distances between members of different species of the same genus (0•053±0•034, 0•060±0•020, and 0•024±0•010, respectively) were in mean smaller than the distances between members of different genera belonging to the same family (0•103±0•053, 0•129±0•051, and 0•044±0•013, respectively). However, the overlap exhibits with standard deviations add to a focus of evidences that some enterobacterial genera are not well defined (Brenner, 1984). In fact, many distances for pairs of species especially belonging to the genera Escherichia, Shigella, Enterobacter, Citrobacter, Klebsiella, and Kluyvera overlap distances for pairs of species belonging to the same genus (FIG. 10). For example, distances for pairs composed by species of Citrobacter and species of Klebsiella overlap distances for pairs composed by two Citrobacter or by two Klebsiella. 
Observing the distance distributions, 16S rDNA distances reveal a clear separation between the families Enterobacteriaceae and Vibrionaceae despite the fact that the family Vibrionaceae is genetically very close to the Enterobacteriaceae (FIGS. 10a and b). Nevertheless, tuf and atpD show higher discriminating power below the family level (FIGS. 10a and b).
There were some discrepancies in the relative distances for the same pairs of taxa between the two genes studied. First, distances between Yersinia species are at least two times lower for atpD than for tuf (FIG. 10c). Also, distances at the family level (between Enterobacteriaceae and Vibrionaceae) show that Enterobacteriaceae is a tightlier knit family with atpD gene (Proteus genus excepted) than with tuf gene. Both genes well delineate taxa belonging to the same species. There is one exception with atpD: Klebsiella planticola and K. ornithinolithica belong to the same genus but fit with taxa belonging to the same species (FIGS. 10a and c). These two species are also very close genotypically with tuf gene. This suggest that Klebsiella planticola and K. ornithinolithica could be two newborn species. tuf and atpD genes exhibit little distances between Escherichia fergusonii and E. coli-Shigella species. Unfortunately, comparison with 16S rDNA could not be achieved because the E. fergusonii 16S rDNA sequence is not yet accessible in GenEMBL database. Therefore, the majority of phenotypically close enterobacteria could be easily discriminated genotypically using tuf and atpD gene sequences.
In conclusion, tuf and atpD genes exhibit phylogenies consistent with 16S rDNA genes phylogeny. For example, they reveal that the family Enterobacteriaceae is monophyletic. Moreover, tuf and atpD distances provide a higher discriminating power than 16S rDNA distances. In fact, tuf and atpD genes discriminate well between different genospecies and are conserved between strains of the same genetic species in such a way that primers and molecular probes for diagnostic purposes could be designed. Preliminary studies support these observations and diagnostic tests based on tuf and atpD sequence data to identify enterobacteria are currently under development.
Example 44
Testing New Pairs of PCR Primers Selected from Two Species-specific Genomic DNA Fragments which are Objects of Our Assigned U.S. Pat. No. 6,001,564.
Objective. The goal of these experiments is to demonstrate that it is relatively easy for a person skilled in the art to find other PCR primer pairs from the species-specific fragments used as targets for detection and identification of a variety of microorganisms. In fact, we wish to prove that the PCR primers previously tested by our group and which are objects of the present patent application are not the only possible good choices for diagnostic purposes. For this example, we used diagnostic targets described in our assigned U.S. Pat. No. 6,001,564.
Experimental strategy. We have selected randomly two species-specific genomic DNA fragments for this experiment. The first one is the 705-bp fragment specific to Staphylococcus epidermidis (SEQ ID NO: 36 from U.S. Pat. No. 6,001,564) while the second one is the 466-bp fragment specific to Moraxella catarrhalis (SEQ ID NO: 29 from U.S. Pat. No. 6,001,564). Subsequently, we have selected from these two fragments a number of PCR primer pairs other than those previously tested. We have chosen 5 new primer pairs from each of these two sequences which are well dispersed along the DNA fragment (FIGS. 11 and 12). We have tested these primers for their specificity and compared them with the original primers previously tested. For the specificity tests, we have tested all bacterial species closely related to the target species based on phylogenetic analysis with three conserved genes (rRNA genes, tuf and atpD). The rational for selecting a restricted number of bacterial species to evaluate the specificity of the new primer pairs is based on the fact that the lack of specificity of a DNA-based assay is attributable to the detection of closely related species which are more similar at the nucleotide level. Based on the phylogenetic analysis, we have selected (i) species from the closely related genus Staphylococcus, Enterococcus, Streptococcus and Listeria to test the specificity of the S. epidermidis-specific PCR assays and (ii) species from the closely related genus Moraxella, Kingella and Neisseria to test the specificity of the M. catarrhalis-specific PCR assays.
Materials and Methods
Bacterial strains. All bacterial strains used for these experiments were obtained from the American Type Culture Collection (ATCC, Rockville, Md.).
Genomic DNA isolation. Genomic DNA was purified from the ATCC reference strains by using the G-nome DNA kit (Bio 101 Inc., Vista, Calif.).
Oligonucleotide design and synthesis. PCR primers were designed with the help of the OligoTM primer analysis software Version 4.0 (National Biosciences Inc., Plymouth, Minn.) and synthesized using a model 391 DNA synthesizer (Applied Biosystems, Foster City, Calif.).
PCR assays. All PCR assays were performed by using genomic DNA purified from reference strains obtained from the ATCC. One μl of purified DNA preparation (containing 0.01 to 1 ng of DNA per μl) was added directly into the PCR reaction mixture. The 20 μL PCR reactions contained final concentrations of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.4 μM of each primer, 200 μM of each of the four dNTPs and 0.5 unit of Taq DNA polymerase (Promega, Madison, Wis.) combined with the TaqStartTM antibody (Clontech Laboratories Inc., Palo Alto, Calif.). An internal control was integrated into all amplification reactions to verify the efficiency of the amplification reaction as well as to ensure that significant PCR inhibition was absent. Primers amplifying a region of 252 bp from a control plasmid added to each amplification reaction were used to provide the internal control. PCR reactions were then subjected to thermal cycling (3 min at 95° C. followed by 30 cycles of 1 second at 95° C. for the denaturation step and 30 seconds at 50 to 65° C. for the annealing-extension step) using a PTC-200 thermal cycler (MJ Research Inc., Watertown, Mass.). PCR amplification products were then analyzed by standard agarose gel (2%) electrophoresis. Amplification products were visualized in agarose gels containing 0.25 μg/mL of ethidium bromide under UV at 254 nm n.
Results
Tables 21 and 22 show the results of specificity tests with the 5 new primer pairs selected from SEQ ID NO: 29 (specific to M. catarrhalis from U.S. Pat. No. 6,001,564) and SEQ ID NO: 36 (specific to S. epidermidis from U.S. Pat. No. 6,001,564), respectively. In order to evaluate the performance of these new primers pairs, we compared them in parallel with the original primer pairs previously tested.
For M. catarrhalis, all of the 5 selected PCR primer pairs were specific for the target species because none of the closely related species could be amplified (Table 21). In fact, the comparison with the original primer pair SEQ ID NO: 118+SEQ ID NO: 119 (from U.S. Pat. No. 6,001,564) revaled that all new pairs showed identical results in terms of specificity and sensitivity thereby suggesting their suitability for diagnostic purposes.
For S. epidermidis, 4 of the 5 selected PCR primer pairs were specific for the target species (Table 22). It should be noted that for 3 of these four primer pairs the annealing temperature had to be increased from 55° C. to 60 or 65° C. to attain specificity for S. epidermidis. Again the comparison with the original primer pair SEQ ID NO: 145+SEQ ID NO: 146 (from U.S. Pat. No. 6,001,564) revealed that these four primer pairs were as good as the original pair. Increasing the annealing temperature for the PCR amplification is well known by persons skilled in the art to be a very effective way to improve the specificity of a PCR assay (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-based Diagnostics in Infectious Disease, Blackwell Scientific Publications, Boston, Mass.). In fact, those skilled in the art are well aware of the fact that the annealing temperature is critical for the optimization of PCR assays. Only the primer pair VBsep3+VBsep4 amplified bacterial species other than S. epidemidis including the staphylococcal species S. capitis, S. cohnii, S. aureus, S. haemolyticus and S. hominis (Table 22). For this non-specific primer pair, increasing the annealing temperature from 55 to 65° C. was not sufficient to attain the desired specificity. One possible explanation for the fact that it appears slightly easier to select species-specific primers for M. catarrhalis than for S. epidermidis is that M. catarrhalis is more isolated in phylogenetic trees than S. epidermidis. The large number of coagulase negative staphylococcal species such as S. epidermidis is largely responsible for this phylogenetic clustering.
Conclusion
These experiment clearly show that it is relatively easy for a person skilled in the art to select, from the species-specific DNA fragments selected as target for identification, PCR primer pairs suitable for diagnostic purposes other than those previously tested. The amplification conditions can be optimize by modifying critical variables such as the annealing temperature to attain the desired specificity and sensitivity. Consequently, we consider that it is legitimate to claim any possible primer sequences selected from the species-specific fragment and that it would be unfair to grant only the claims dealing with the primer pairs previously tested. By extrapolation, these results strongly suggest that it is also relatively easy for a person skilled in the art to select, from the species-specific DNA fragments, DNA probes suitable for diagnostic purposes other than those previously tested.
Example 45
Testing Modified Versions of PCR Primers Derived from the Sequence of Several Primers which are Objects of U.S. Pat. No. 6,001,564.
Objective. The purpose of this project is to verify the efficiency of amplification by modified PCR primers derived from primers previously tested. The types of primer modifications to be tested include (i) variation of the sequence at one or more nucleotide positions and (ii) increasing or reducing the length of the primers. For this example, we used diagnostic targets described in U.S. Pat. No. 6,001,564.
Experimental Strategy:
Testing Primers with Nucleotide Changes
We have designed 13 new primers which are derived from the S. epidermidis-specific SEQ ID NO: 146 from U.S. Pat. No. 6,001,564 (Table 23). These primers have been modified at one or more nucleotide positions. As shown in Table 23, the nucleotide changes were introduced all along the primer sequence. Furthermore, instead of modifying the primer at any nucleotide position, the nucleotide changes were introduced at the third position of each codon to better reflect potential genetic variations in vivo. It should be noted that no nucleotide changes were introduced at the 3′ end of the oligonucleotide primers because those skilled in the art are well aware of the fact that mismatches at the 3′ end should be avoided. (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). All of these modified primers were tested in PCR assays in combination with SEQ ID NO: 145 from U.S. Pat. No. 6,001,564 and the efficiency of the amplification was compared with the original primer pair SEQ ID NO: 145+SEQ ID NO: 146 previously tested in U.S. Pat. No. 6,001,564.
Testing Shorter or Longer Versions of Primers
We have designed shorter and longer versions of the original S. epidermidis-specific PCR primer pair SEQ ID NO: 145+146 from U.S. Pat. No. 6,001,564 (Table 24) as well as shorter versions of the original P. aeruginosa-specific primer pair SEQ ID NO: 83+84 from U.S. Pat. No. 6,001,564 (Table 25). As shown in Tables 24 and 25, both primers of each pair were shortened or lengthen to the same length. Again, those skilled in the art know that the melting temperature of both primers from a pair should be similar to avoid preferential binding at one primer binding site which is detrimental in PCR (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-based Diagnostics in Infectious Disease, Blackwell Scientific Publications, Boston, Mass.). All of these shorter or longer primer versions were tested in PCR assays and the efficiency of the amplification was compared with the original primer pair SEQ ID NOs 145 and 146.
Materials and Methods
See the Materials and methods section of Example 44.
Results
Testing Primers with Nucleotide Changes
The results of the PCR assays with the 13 modified versions of SEQ ID NO: 146 from U.S. Pat. No. 6,001,564 are shown in Table 23. The 8 modified primers having a single nucleotide variation showed an efficiency of amplification identical to the original primer pair based on testing with 3 different dilutions of genomic DNA. The four primers having two nucleotide variations and primer VBmut12 having 3 nucleotide changes also showed PCR results identical to those obtained with the original pair. Finally, primer VBmut13 with four nucleotide changes showed a reduction in sensitivity by approximately one log as compared with the original primer pair. However, reducing the annealing temperature from 55 to 50° C. gave an efficiency of amplification very similar to that observed with the original primer pair (Table 23). In fact, reducing the annealing temperature of PCR cycles represents an effective way to reduce the stringency of hybridization for the primers and consequently allows the binding of probes with mismatches (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). Subsequently, we have confirmed the specificity of the PCR assays with each of these 13 modified versions of SEQ ID NO: 146 from U.S. Pat. No. 6,001,564 by performing amplifications from all bacterial species closely related to S. epidermidis which are listed in Table 22.
Testing Shorter or Longer Versions of Primers
For these experiments, two primer pairs were selected: i) SEQ ID NO: 145+146 from U.S. Pat. No. 6,001,564 (specific to S. epidermidis) which are AT rich and ii) SEQ ID NO: 83+84 (specific to P. aeruginosa) which are GC rich. For the AT rich sequence, primers of 15 to 30 nucleotide in length were designed (Table 24) while for the GC rich sequences, primers of 13 to 19 nucleotide in length were designed (Table 25).
Table 24 shows that, for an annealing temperature of 55° C., the 30- 25-, 20- and 17-nucleotide versions of SEQ ID NO: 145 and 146 from U.S. Pat. No. 6,001,564 all showed identical results as compared with the original primer pair except that the 17-nucleotide version amplified slightly less efficiently the S. epidermidis DNA. Reducing the annealing temperature from 55 to 45° C. for the 17-nucleotide version allowed to increase the amplification efficiency to a level very similar to that with the original primer pair (SEQ ID NO: 145+146 from U.S. Pat. No. 6,001,564). Regarding the 15-nucleotide version, there was amplification of S. epidermidis DNA only when the annealing temperature was reduced to 45° C. Under those PCR conditions the assay remained S. epidermidis-specific but the amplification signal with S. epidermidis DNA was slightly lower as compared with the original primer pair. Subsequently, we have further confirmed the specificity of the shorter or longer versions by amplifying DNA from all bacterial species closely related to S. epidermidis which are listed in Table 22.
Table 25 shows that, for an annealing temperature of 55° C., all shorter versions of SEQ ID NO: 83 and 84 from U.S. Pat. No. 6,001,564 showed identical PCR results as compared with the original primer pair. As expected, these results show that it is simpler to reduce the length of GC rich as compared with AT rich. This is attributable to the fact that GC binding is more stable than AT binding.
Conclusion
Testing Primers with Nucleotide Changes
The above experiments clearly show that PCR primers may be modified at one or more nucleotide positions without affecting the specificity and the sensitivity of the PCR assay. These results strongly suggest that a given oligonucleotide can detect variant genomic sequences from the target species. In fact, the nucleotide changes in the selected primers were purposely introduced at the third position of each codon to mimic nucleotide variation in genomic DNA. Thus we conclude that it is justified to claim “a variant thereof” for i) the SEQ IDs of the fragments and oligonucleotides which are object of the present patent application and ii) genomic variants of the target species.
Testing Shorter or Longer Versions of Primers
The above experiments clearly show that PCR primers may be shorter or longer without affecting the specificity and the sensitivity of the PCR assay. We have showed that oligonucleotides ranging in sizes from 13 to 30 nucleotides may be as specific and sensitive as the original primer pair from which they were derived. Consequently, these results suggest that it is not exaggerated to claim sequences having at least 12 nucleotide in length.
This invention has been described herein above, and it is readily apparent that modifications can be made thereto without departing from the spirit of this invention. These modifications are under the scope of this invention, as defined in the appended claims.







TABLE 1







Distribution (%) of nosocomial pathogens for various


human infections in USA (1990-1992)1.












Pathogen
UTI2
SSI3
BSI4
Pneumonia
CSF5















Escherichia coli
27
9
5
4
2


Staphylococcus aureus
2
21
17
21
2


Staphylococcus epidermidis
2
6
20
0
1


Enterococcus faecalis
16
12
9
2
0


Enterococcus faecium
1
1
0
0
0


Pseudomonas aeruginosa
12
9
3
18
0


Klebsiella pneumoniae
7
3
4
9
0


Proteus mirabilis
5
3
1
2
0


Streptococcus pneumoniae
0
0
3
1
18


Group B Streptococci
1
1
2
1
6


Other treptococci
3
5
2
1
3


Haemophilus influenzae
0
0
0
6
45


Neisseria meningitidis
0
0
0
0
14


Listeria monocytogenes
0
0
0
0
3


Other enterococci
1
1
0
0
0


Other staphylococci
2
8
13
2
0


Candida albicans
9
3
5
5
0


Other Candida
2
1
3
1
0


Enterobacter sp.
5
7
4
12
2


Acinetobacter sp.
1
1
2
4
2


Citrobacter sp.
2
1
1
1
0


Serratia marcescens
1
1
1
3
1


Other klebsiella
1
1
1
2
1


Others
0
6
4
5
0





1Data recorded by the National Nosocomial Infections Surveillance (NNIS) from 80 hospitals (Emori and Gaynes, 1993, Clin. Microbiol. Rev., 6: 428-442).


2Urinary tract infection.


3Surgical site infection.


4Bloodstream infection.


5Cerebrospinal fluid.













TABLE 2







Distribution (%) of bloodstream infection pathogens in Quebec


(1995), Canada (1992), UK (1969-1988) and USA (1990-1992).










UK3
















Com-

USA4





munity-
Hospital-
Hospital-


Organism
Quebec1
Canada2
acquired
acquired
acquired















E. coli
15.6 
53.8 
24.8
20.3
5.0


S. epidermidis
25.8
—
0.5
7.2
31.0 


and other


CoNS5


S. aureus
9.6
—
9.7
19.4
16.0 


S. pneumoniae
6.3
—
22.5
2.2
—


E. faecalis
3.0
—
1.0
4.2
—


E. faecium
2.6
—
0.2
0.5
—


Enterococcus
—
—
—
—
9.0


sp.


H. influenzae
1.5
—
3.4
0.4
—


P. aeruginosa
1.5
8.2
1.0
8.2
3.0


K. pneumoniae
3.0
11.2 
3.0
9.2
4.0


P. mirabilis
—
3.9
2.8
5.3
1.0


S. pyogenes
—
—
1.9
0.9
—


Enterobacter
4.1
5.5
0.5
2.3
4.0


sp.


Candida sp.
8.5
—
—
1.0
8.0


Others
18.5 
17.4 
28.7
18.9
19.0 





1Data obtained for 270 isolates collected at the Centre Hospitalier de I'Université Laval (CHUL) during a 5 month period (May to October 1995).


2Data from 10 hospitals throughout Canada representing 941 gram-negative isolates. (Chamberland et al., 1992, Clin. Infect. Dis., 15: 615-628).


3Data from a 20-year study (1969-1988) for nearly 4000 isolates. (Eykyn et al., 1990, J. Antimicrob. Chemother., Suppl. C, 25: 41-58).


4Data recorded by the National Nosocomial Infections Surveillance (NNIS) from 80 hospitals (Emori and Gaynes, 1993, Clin. Microbiol. Rev., 6: 428-442).


5Coagulase-negative staphylococci.













TABLE 3







Distribution of positive and negative clinical specimens tested at the


microbiology laboratory of the CHUL (February 1994-January 1995).













% of


Clinical specimens
No. of samples
% of positive
negative


and/or sites
tested (%)
specimens
specimens













Urine
17,981 (54.5)  
19.4
80.6


Blood culture/marrow
10,010 (30.4)  
6.9
93.1


Sputum
1,266 (3.8)  
68.4
31.6


Superficial pus
1,136 (3.5)  
72.3
27.7


Cerebrospinal fluid
553 (1.7)
1.0
99.0


Synovial fluid
523 (1.6)
2.7
97.3


Respiratory tract
502 (1.5)
56.6
43.4


Deep pus
473 (1.4)
56.8
43.2


Ears
289 (0.9)
47.1
52.9


Pleural and pericardial fluid
132 (0.4)
1.0
99.0


Peritoneal fluid
101 (0.3)
28.6
71.4


Total:
32,966 (100.0) 
20.0
80.0
















TABLE 4





Example of microbial species for which tuf and/or atpD and/or recA


nucleic acids and/or sequences are used in the present invention.







Bacterial species









Abiotrophia adiacens



Abiotrophia defectiva



Achromobacter xylosoxidans subsp. denitrificans



Acetobacterium woodi



Acetobacter aceti



Acetobacter altoacetigenes



Acetobacter polyoxogenes



Acholeplasma laidlawii



Acidothermus cellulolyticus



Acidiphilum facilis



Acinetobacter baumannii



Acinetobacter calcoaceticus



Acinetobacter lwoffii



Actinomyces meyeri



Aerococcus viridans



Aeromonas hydrophila



Aeromonas salmonicida



Agrobacterium radiobacter



Agrobacterium tumefaciens



Alcaligenes faecalis subsp. faecalis



Allochromatium vinosum



Anabaena variabilis



Anacystis nidulans



Anaerorhabdus furcosus



Aquifex aeolicus



Aquifex pyrophilus



Arcanobacterium haemolyticum



Archaeoglobus fulgidus



Azotobacter vinelandii



Bacillus anthracis



Bacillus cereus



Bacillus firmus



Bacillus halodurans



Bacillus megaterium



Bacillus mycoides



Bacillus pseudomycoides



Bacillus stearothermophilus



Bacillus subtilis



Bacillus thuringiensis



Bacillus weihenstephanensis



Bacteroides distasonis



Bacteroides fragilis



Bacteroides forsythus



Bacteroides ovatus



Bacteroides vulgatus



Bartonella henselae



Bifidobacterium adolescentis



Bifidobacterium breve



Bifidobacterium dentium



Bifidobacterium longum



Blastochloris viridis



Borrelia burgdorferi



Bordetella pertussis



Bordetella bronchiseptica



Brucella abortus



Brevibacterium linens



Brevibacterium flavum



Brevundimonas diminuta



Buchnera aphidicola



Budvicia aquatica



Burkholderia cepacia



Burkholderia mallei



Burkholderia pseudomallei



Buttiauxella agrestis



Butyrivibrio fibrisolvens



Campylobacter coli



Campylobacter curvus



Campylobacter fetus subsp. fetus



Campylobacter fetus subsp. venerealis



Campylobacter gracilis



Campylobacter jejuni



Campylobacter jejuni subsp. doylei



Campylobacter jejuni subsp. jejuni



Campylobacter lari



Campylobacter rectus



Campylobacter sputorum subsp. sputorum



Campylobacter upsaliensis



Cedecea davisae



Cedecea lapagei



Cedecea neteri



Chlamydia pneumoniae



Chlamydia psittaci



Chlamydia trachomatis



Chlorobium vibrioforme



Chloroflexus aurantiacus



Chryseobacterium meningosepticum



Citrobacter amalonaticus



Citrobacter braakii



Citrobacter farmeri



Citrobacter freundii



Citrobacter koseri



Citrobacter sedlakii



Citrobacter werkmanii



Citrobacter youngae



Clostridium acetobutylicum



Clostridium beijerinckii



Clostridium bifermentans



Clostridium botulinum



Clostridium difficile



Clostridium innocuum



Clostridium histolyticum



Clostridium novyi



Clostridium septicum



Clostridium perfringens



Clostridium ramosum



Clostridium tertium



Clostridium tetani



Comamonas acidovorans



Corynebacterium accolens



Corynebacterium bovis



Corynebacterium cervicis



Corynebacterium diphtheriae



Corynebacterium flavescens



Corynebacterium genitalium



Corynebacterium glutamicum



Corynebacterium jeikeium



Corynebacterium kutscheri



Corynebacterium minutissimum



Corynebacterium mycetoides



Corynebacterium pseudodiphtheriticum



Corynebacterium pseudogenitalium



Corynebacterium pseudotuberculosis



Corynebacterium renale



Corynebacterium striatum



Corynebacterium ulcerans



Corynebacterium urealyticum



Corynebacterium xerosis



Coxiella burnetii



Cytophaga lytica



Deinococcus radiodurans



Deinonema sp.



Edwardsiella hoshinae



Edwardsiella tarda



Ehrlichia canis



Ehrlichia risticii



Eikenella corrodens



Enterobacter aerogenes



Enterobacter agglomerans



Enterobacter amnigenus



Enterobacter asburiae



Enterobacter cancerogenus



Enterobacter cloacae



Enterobacter gergoviae



Enterobacter hormaechei



Enterobacter sakazakii



Enterococcus avium



Enterococcus casseliflavus



Enterococcus cecorum



Enterococcus columbae



Enterococcus dispar



Enterococcus durans



Enterococcus faecalis



Enterococcus faecium



Enterococcus flavescens



Enterococcus gallinarum



Enterococcus hirae



Enterococcus malodoratus



Enterococcus mundtii



Enterococcus pseudoavium



Enterococcus raffinosus



Enterococcus saccharolyticus



Enterococcus solitarius



Enterococcus sulfureus



Clostridium sordellii



Erwinia amylovora



Erwinia carotovora



Escherichia coli



Escherichia fergusonii



Escherichia hermannii



Escherichia vulneris



Eubacterium lentum



Eubacterium nodatum



Ewingella americana



Francisella tularensis



Frankia alni



Fervidobacterium islandicum



Fibrobacter succinogenes



Flavobacterium ferrigeneum



Flexistipes sinusarabici



Fusobacterium gonidiaformans



Fusobacterium necrophorum subsp. necrophorum



Fusobacterium nucleatum subsp. polymorphum



Gardnerella vaginalis



Gemella haemolysans



Gemella morbillorum



Globicatella sanguis



Gloeobacter violaceus



Gloeothece sp.



Gluconobacter oxydans



Haemophilus actinomycetemcomitans



Haemophilus aphrophilus



Haemophilus ducreyi



Haemophilus haemolyticus



Haemophilus influenzae



Haemophilus parahaemolyticus



Haemophilus parainfluenzae



Haemophilus paraphrophilus



Haemophilus segnis



Hafnia alvei



Halobacterium marismortui



Halobacterium salinarum



Haloferax volcanii



Helicobacter pylori



Herpetoshiphon aurantiacus



Kingella kingae



Klebsiella ornithinolytica



Klebsiella oxytoca



Klebsiella planticola



Klebsiella pneumoniae subsp. ozaenae



Klebsiella pneumoniae subsp. pneumoniae



Klebsiella pneumoniae subsp. rhinoscleromatis



Klebsiella terrigena



Kluyvera ascorbata



Kluyvera cryocrescens



Kluyvera georgiana



Kocuria kristinae



Lactobacillus acidophilus



Lactobacillus garvieae



Lactobacillus paracasei



Lactobacillus casei subsp. casei



Lactococcus garvieae



Lactococcus lactis



Lactococcus lactis subsp. lactis



Legionella micdadei



Legionella pneumophila subsp. pneumophila



Leminorella grimontii



Leminorella richardii



Leptospira biflexa



Leptospira interrogans



Leuconostoc mesenteroides subsp. dextranicum



Listeria innocua



Listeria ivanovii



Listeria monocytogenes



Listeria seeligeri



Macrococcus caseolyticus



Magnetospirillum magnetotacticum



Megamonas hypermegale



Methanobacterium thermoautotrophicum



Methanococcus jannaschii



Methanococcus vannielii



Methanosarcina barkeri



Methanosarcina jannaschii



Methylobacillus flagellatum



Methylomonas clara



Micrococcus luteus



Micrococcus lylae



Mitsuokella multacidus



Mobiluncus curtisii subsp. holmesii



Moellerella thermoacetica



Moellerella wisconsensis



Moorella thermoacetica



Moraxella catarrhalis



Moraxella osloensis



Morganella morganii subsp. morganii



Mycobacterium avium



Mycobacterium bovis



Mycobacterium gordonae



Mycobacterium kansasii



Mycobacterium leprae



Mycobacterium terrae



Mycobacterium tuberculosis



Mycoplasma capricolum



Mycoplasma gallisepticum



Mycoplasma genitalium



Mycoplasma hominis



Mycoplasma pirum



Mycoplasma mycoides



Mycoplasma pneumoniae



Mycoplasma pulmonis



Mycoplasma salivarium



Myxococcus xanthus



Neisseria animalis



Neisseria canis



Neisseria cinerea



Neisseria cuniculi



Neisseria elongata subsp. elongata



Neisseria elongata subsp. intermedia



Neisseria flava



Neisseria flavescens



Neisseria gonorrhoeae



Neisseria lactamica



Leclercia adecarboxylata



Neisseria meningitidis



Neisseria mucosa



Neisseria perflava



Neisseria pharyngis var. flava



Neisseria polysaccharea



Neisseria sicca



Neisseria subflava



Neisseria weaveri



Obesumbacterium proteus



Ochrobactrum anthropi



Pantoea agglomerans



Pantoea dispersa



Paracoccus denitrificans



Pasteurella multocida



Pectinatus frisingensis



Peptococcus niger



Peptostreptococcus anaerobius



Peptostreptococcus asaccharolyticus



Peptostreptococcus prevotii



Phormidium ectocarpi



Pirellula marina



Planobispora rosea



Plesiomonas shigelloides



Plectonema boryanum



Porphyromonas asaccharolytica



Porphyromonas gingivalis



Pragia fontium



Prevotella buccalis



Prevotella melaninogenica



Prevotella oralis



Prevotella ruminocola



Prochlorothrix hollandica



Propionibacterium acnes



Propionigenium modestum



Proteus mirabilis



Proteus penneri



Proteus vulgaris



Providencia alcalifaciens



Providencia rettgeri



Providencia rustigianii



Providencia stuartii



Pseudomonas aeruginosa



Pseudomonas fluorescens



Pseudomonas putida



Pseudomonas stutzeri



Psychrobacter phenylpyruvicum



Pyrococcus abyssi



Rahnella aquatilis



Rickettsia prowazekii



Rhizobium leguminosarum



Rhizobium phaseoli



Rhodobacter capsulatus



Rhodobacter sphaeroides Rhodopseudomonas



palustris



Rhodospirillum rubrum



Ruminococcus albus



Ruminococcus bromii



Salmonella bongori



Salmonella choleraesuis subsp. arizonae



Salmonella choleraesuis subsp choleraesuis



Salmonella choleraesuis subsp. diarizonae



Salmonella choleraesuis subsp. houtenae



Salmonella choleraesuis subsp. indica



Salmonella choleraesuis subsp. salamae



Serpulina hyodysenteriae



Serratia ficaria



Serratia fonticola



Serratia grimesii



Serratia liquefaciens



Serratia marcescens



Serratia odorifera



Serratia plymuthica



Serratia rubidaea



Shewanella putrefaciens



Shigella boydii



Shigella dysenteriae



Shigella flexneri



Shigella sonnei



Sinorhizobium meliloti



Spirochaeta aurantia



Staphylococcus aureus



Staphylococcus aureus subsp. aureus



Staphylococcus auricularis



Staphylococcus capitis subsp. capitis



Staphylococcus cohnii subsp. cohnii



Staphylococcus epidermidis



Staphylococcus haemolyticus



Staphylococcus hominis



Staphylococcus hominis subsp. hominis



Staphylococcus lugdunensis



Staphylococcus saprophyticus



Staphylococcus sciuri subsp. sciuri



Staphylococcus simulans



Staphylococcus warneri



Stigmatella aurantiaca



Stenotrophomonas maltophilia



Streptococcus acidominimus



Streptococcus agalactiae



Streptococcus anginosus



Streptococcus bovis



Streptococcus cricetus



Streptococcus cristatus



Streptococcus downei



Streptococcus dysgalactiae



Streptococcus equi subsp. equi



Streptococcus ferus



Streptococcus gordonii



Streptococcus macacae



Streptococcus mitis



Streptococcus mutans



Streptococcus oralis



Streptococcus parasanguinis



Streptococcus pneumoniae



Streptococcus pyogenes



Streptococcus ratti



Streptococcus salivarius



Streptococcus salivarius subsp. thermophilus



Streptococcus sanguinis



Streptococcus sobrinus



Streptococcus suis



Streptococcus uberis



Streptococcus vestibularis



Streptomyces anbofaciens



Streptomyces aureofaciens



Streptomyces cinnamoneus



Streptomyces coelicolor



Streptomyces collinus



Streptomyces lividans



Streptomyces netropsis



Streptomyces ramocissimus



Streptomyces rimosus



Streptomyces venezuelae



Succinivibrio dextrinosolvens



Synechococcus sp.



Synechocystis sp.



Tatumella ptyseos



Taxeobacter occealus



Tetragenococcus halophilus



Thermoplasma acidophilum



Thermotoga maritima



Thermus aquaticus



Thermus thermophilus



Thiobacillus ferrooxidans



Thiomonas cuprina



Trabulsiella guamensis



Treponema pallidum



Ureaplasma urealyticum



Veillonella parvula



Vibrio alginolyticus



Vibrio anguillarum



Vibrio cholerae



Vibrio mimicus



Wolinella succinogenes



Xanthomonas citri



Xanthomonas oryzae



Xenorhabdus bovieni



Xenorhabdus nematophilus



Yersinia bercovieri



Yersinia enterocolitica



Yersinia frederiksensii



Yersinia intermedia



Yersinia pestis



Yersinia pseudotuberculosis



Yersinia rohdei



Yokenella regensburgei



Zoogloea ramigera







Fungal species









Absidia corymbifera



Absidia glauca



Alternaria alternata



Arxula adeninivorans



Aspergillus flavus



Aspergillus fumigatus



Aspergillus nidulans



Aspergillus niger



Aspergillus oryzae



Aspergillus terreus



Aspergillus versicolor



Aureobasidium pullulans



Basidiobolus ranarum



Bipolaris hawaiiensis



Bilophila wadsworthia



Blastoschizomyces capitatus



Blastomyces dermatitidis



Candida albicans



Candida catenulata



Candida dubliniensis



Candida famata



Candida glabrata



Candida guilliermondii



Candida haemulonii



Candida inconspicua



Candida kefyr



Candida krusei



Candida lambica



Candida lusitaniae



Candida norvegica



Candida norvegensis



Candida parapsilosis



Candida rugosa



Candida sphaerica



Candida tropicalis



Candida utilis



Candida viswanathii



Candida zeylanoides



Cladophialophora carrionii



Coccidioides immitis



Coprinus cinereus



Cryptococcus albidus



Cryptococcus humicolus



Cryptococcus laurentii



Cryptococcus neoformans



Cunninghamella bertholletiae



Curvularia lunata



Emericella nidulans



Emmonsia parva



Eremothecium gossypii



Exophiala dermatitidis



Exophiala jeanselmei



Exophiala moniliae



Exserohilum rostratum



Eremothecium gossypii



Fonsecaea pedrosoi



Fusarium moniliforme



Fusarium oxysporum



Fusarium solani



Geotrichum sp.



Histoplasma capsulatum



Hortaea werneckii



Issatchenkia orientalis Kudrjanzev



Kluyveromyces lactis



Malassezia furfur



Malassezia pachydermatis



Malbranchea filamentosa



Metschnikowia pulcherrima



Microsporum audouinii



Microsporum canis



Mucor circinelloides



Neurospora crassa



Paecilomyces lilacinus



Paracoccidioides brasiliensis



Penicillium marneffei



Phialaphora verrucosa



Pichia anomala



Piedraia hortai



Podospora anserina



Podospora curvicolla



Puccinia graminis



Pseudallescheria boydii



Reclinomonas americana



Rhizomucor racemosus



Rhizopus oryzae



Rhodotorula minuta



Rhodotorula mucilaginosa



Saccharomyces cerevisiae



Saksenaea vasiformis



Schizosaccharomyces pombe



Scopulariopsis koningii



Sordaria macrospora



Sporobolomyces salmonicolor



Sporothrix schenckii



Stephanoascus ciferrii



Syncephalastrum racemosum



Trichoderma reesei



Trichophyton mentagrophytes



Trichophyton rubrum



Trichophyton tonsurans



Trichosporon cutaneum



Ustilago maydis



Wangiella dermatitidis



Yarrowia lipolytica







Parasitical species









Babesia bigemina



Babesia bovis



Babesia microti



Blastocystis hominis



Crithidia fasciculata



Cryptosporidium parvum



Entamoeba histolytica



Giardia lamblia



Kentrophoros sp.



Leishmania aethiopica



Leishmania amazonensis



Leishmania braziliensis



Leishmania donovani



Leishmania infantum



Leishmania enriettii



Leishmania gerbilli



Leishmania guyanensis



Leishmania hertigi



Leishmania major



Leishmania mexicana



Leishmania panamensis



Leishmania tarentolae



Leishmania tropica



Neospora caninum



Onchocerca volvulus



Plasmodium berghei



Plasmodium falciparum



Plasmodium knowlesi



Porphyra purpurea



Toxoplasma gondii



Treponema pallidum



Trichomonas tenax



Trichomonas vaginalis



Trypanosoma brucei



Trypanosoma brucei subsp. brucei



Trypanosoma congolense



Trypanosoma cruzi

















TABLE 5







Antimicrobial agents resistance genes selected for diagnostic purposes











Gene
Antimicrobial agent
Bacteria1
ACCESSION NO.
SEQ ID NO.





aac(3)-Ib2
Aminoglycosides
Enterobacteriaceae
L06157





Pseudomonads


aac(3)-IIb2
Aminoglycosides
Enterobacteriaceae,
M97172




Pseudomonads


aac(3)-IVa2
Aminoglycosides
Enterobacteriaceae
X01385


aac(3)-VIa2
Aminoglycosides
Enterobacteriaceae,
M88012




Pseudomonads


aac(2′)-1a2
Aminoglycosides
Enterobacteriaceae,
X04555




Pseudomonads


aac(6′)-aph(2′′)2
Aminoglycosides
Enterococcus sp.,

83-863




Staphylococcus sp.


aac(6′)-Ia,2
Aminoglycosides
Enterobacteriaceae,
M18967




Pseudomonads


aac(6′)-Ic2
Aminoglycosides
Enterobacteriaceae,
M94066




Pseudomonads


aac(6′)-IIa2
Aminoglycosides
Pseudomonads

1124


aadB [ant(2′′)-Ia2]
Aminoglycosides
Enterobacteriaceae

53-543


aacC1 [aac(3)-Ia2]
Aminoglycosides
Pseudomonads

55-563


aacC2 [aac(3)-IIa2]
Aminoglycosides
Pseudomonads

57-583


aacC3 [aac(3)-III2]
Aminoglycosides
Pseudomonads

59-603


aacA4 [aac(6′)-Ib2]
Aminoglycosides
Pseudomonads

65-663


ant(3′′)-Ia2
Aminoglycosides
Enterobacteriaceae,
X02340




Enterococcus sp.,
M10241




Staphylococcus sp.


ant(4′)-Ia2
Aminoglycosides
Staphylococcus sp.
V01282


aph(3′)-Ia2
Aminoglycosides
Enterobacteriaceae,
J01839




Pseudomonads


aph(3′)-IIa2
Aminoglycosides
Enterobacteriaceae,
V00618




Pseudomonads


aph(3′)-IIIa2
Aminoglycosides
Enterococcus sp.,
V01547




Staphylococcus sp.


aph(3′)-VIa2
Aminoglycosides
Enterobacteriaceae,
X07753




Pseudomonads


rpsL2
Streptomycin
M. tuberculosis,
X80120




M. avium complex
U14749





X70995





L08011


blaOXA5,6
β-lactams
Enterobacteriaceae,
Y10693
1104




Pseudomonads
AJ238349





AJ009819





X06046





X03037





X07260





U13880





X75562





AF034958





J03427





Z22590





U59183





L38523





U63835





AF043100





AF060206





U85514





AF043381





AF024602





AF064820


blaROB5
β-lactams
Haemophilus sp.

45-483


blaSHV5,6
β-lactams
Enterobacteriacea,
AF124984
41-443




Pseudomonas aeruginosa
AF148850





M59181





X98099





M33655





AF148851





X53433





L47119





AF074954





X53817





AF096930





X55640





Y11069





U20270





U92041





S82452





X98101





X98105





AF164577





AJ011428





AF116855





AB023477





AF293345





AF227204





AF208796





AF132290


blaTEM5,6
β-lactams
Enterobacteriaceae,
AF012911
37-403




Neisseria sp.,
U48775




Haemophilus sp.
AF093512





AF052748





X64523





Y13612





X57972





AF157413





U31280





U36911





U48775





V00613





X97254





AJ012256





X04515





AF126482





U09188





M88143





Y14574





AF188200





AJ251946





Y17581





Y17582





Y17583





M88143





U37195





Y17584





X64523





U95363





Y10279





Y10280





Y10281





AF027199





AF104441





AF104442





AF062386





X57972





AF047171





AF188199





AF157553





AF190694





AF190695





AF190693





AF190692


blaSHV5,6
β-lactams
Enterobacteriacea,
AF124984
41-443




Pseudomonas aeruginosa
AF148850





M59181





X98099





M33655





AF148851





X53433





L47119





AF074954





X53817





AF096930





X55640





Y11069





U20270





U92041





S82452





X98101





X98105





AF164577





AJ011428





AF116855





AB023477





AF293345





AF227204





AF208796





AF132290


blaTEM5,6
β-lactams
Enterobacteriaceae,
AF012911
37-403




Neisseria sp.,
U48775




Haemophilus sp.
AF093512





AF052748





X64523





Y13612





X57972





AF157413





U31280





U36911





U48775





V00613





X97254





AJ012256





X04515





AF126482





U09188





M88143





Y14574





AF188200





AJ251946





Y17581





Y17582





Y17583





M88143





U37195





Y17584





X64523





U95363





Y10279





Y10280





Y10281





AF027199





AF104441





AF104442





AF062386





X57972





AF047171





AF188199





AF157553





AF190694





AF190695





AF190693





AF190692


blaCARB5
β-lactams
Pseudomonas sp.,
J05162




Enterobacteriaceae
S46063





M69058





U14749





D86225





D13210





Z18955





AF071555





AF153200





AF030945


blaCTX-M-15
β-lactams
Enterobacteriaceae
X92506


blaCTX-M-25
β-lactams
Enterobacteriaceae
X92507


blaCMY-21
β-lactams
Enterobacteriaceae
X91840





AJ007826





AJ011293





AJ011291





Y17716





Y16783





Y16781





Y15130





U77414





S83226





Y15412





X78117


blaIMP5
β-lactams
Enterobacteriaceae,
AJ223604




Pseudomonas aeruginosa
S71932





D50438





D29636





X98393





AB010417





D78375


blaPER-15
β-lactams
Enterobacteriaceae,
Z21957




Pseudomodanaceae


blaPER-27
β-lactams
Enterobacteriaceae
X93314


blaZ12
β-lactams
Enterococcus sp.,

1114




Staphylococcus sp.


mecA12
β-lactams
Staphylococcus sp.

97-983


pbp1a13
β-lactams
Streptococcus pneumoniae
M90527
1004-1018,





X67872
1648, 2056-2064,





AB006868
2273-2276





AB006874





X67873





AB006878





AB006875





AB006877





AB006879





AF046237





AF046235





AF026431





AF046232





AF046233





AF046236





X67871





Z49095





AF046234





AB006873





X67866





X67868





AB006870





AB006869





AB006872





X67870





AB006871





X67867





X67869





AB006876





AF046230





AF046238





Z49094


pbp2b13
β-lactams
Streptococcus pneumoniae
X16022
1019-1033





M25516





M25518





M25515





U20071





U20084





U20082





U20067





U20079





Z22185





U20072


pbp2b13
β-lactams
Streptococcus pneumoniae
U20083





U20081





M25522





U20075





U20070





U20077





U20068





Z22184





U20069





U20078





M25521





M25525





M25519





Z21981





M25523





M25526





U20076





U20074





M25520





M25517





M25524





Z22230





U20073





U20080


pbp2x13
β-lactams
Streptococcus pneumoniae
X16367
1034-1048





X65135





AB011204





AB011209





AB011199





AB011200





AB011201





AB011202





AB011198





AB011208





AB011205





AB015852





AB011210





AB015849





AB015850





AB015851





AB015847





AB015846





AB011207





AB015848





Z49096


int
β-lactams,
Enterobacteriaceae,

99-1023



trimethoprim


sul
aminoglycosides,
Pseudomonads

103-1063



antiseptic,



chloramphenicol


ermA14
Macrolides,
Staphylococcus sp.

1134



lincosamides,



streptogramin B


ermB14
Macrolides,
Enterobacteriaceae,

1144



lincosamides,
Staphylococcus sp.



streptogramin B
Enterococcus sp.




Streptococcus sp.


ermC14
Macrolides,
Enterobacteriaceae,

1154



lincosamides,
Staphylococcus sp.



streptogramin B


ereA12
Macrolides
Enterobacteriaceae,
M11277




Staphylococcus sp.
E01199





AF099140


ereB12
Macrolides
Enterobacteriaceae
A15097




Staphylococcus sp.
X03988


msrA12
Macrolides
Staphylococcus sp.

77-803


mefA, mefE8
Macrolides
Streptococcus sp.
U70055





U83667


mphA8
Macrolides
Enterobacteriaceae,
D16251




Staphylococcus sp.
U34344





U36578


linA/linA′9
Lincosamides
Staphylococcus sp.
J03947





M14039





A15070





E01245


linB10
Lincosamides
Enterococcus faecium
AF110130





AJ238249


vga15
Streptrogramin
Staphylococcus sp.
M90056
89-903





U82085


vgb15
Streptrogramin
Staphylococcus sp.
M36022





M20219





AF015628


vat15
Streptrogramin
Staphylococcus sp.
L07778
87-883


vatB15
Streptrogramin
Staphylococcus sp.
U19459





L38809


satA15
Streptrogramin
Enterococcus faecium
L12033
81-823


mupA12
Mupirocin
Staphylococcus aureus
X75439





X59478





X59477


gyrA16
Quinolones
Gram-positive and
X95718
1255, 1607-1608,




gram-negative bacteria
X06744
1764-1776,





X57174
2013-2014,





X16817
2277-2280





X71437





AF065152





AF060881





D32252


parC/grlA16
Quinolones
Gram-positive and
AB005036
1777-1785




gram-negative bacteria
AF056287





X95717





AF129764





AB017811





AF065152


parE/grlB16
Quinolones
Gram-positive bacteria
X95717





AF065153





AF058920


norA16
Quinolones
Staphylococcus sp.
D90119





M80252





M97169


mexR (nalB)16
Quinolones
Pseudomonas aeruginosa
U23763


nfxB16
Quinolones
Pseudomonas aeruginosa
X65646


cat12
Chloramphenicol
Gram-positive and
M55620




gram-negative bacteria
X15100





A24651





M28717





A00568





A00569





X74948





Y00723





A24362





A00569





M93113





M62822





M58516





V01277





X02166





M77169





X53796





J01841





X07848


ppflo-like
Chloramphenicol

AF071555


embB17
Ethambutol
Mycobacterium tuberculosis
U68480


pncA17
Pyrazinamide
Mycobacterium tuberculosis
U59967


rpoB17
Rifampin
Mycobacterium tuberculosis
AF055891





AF055892





S71246





L27989





AF055893


inhA17
Isoniazid
Mycobacterium tuberculosis
AF106077





U02492


vanA12
Vancomycin
Enterococcus sp.

67-703






1049-1057


vanB12
Vancomycin
Enterococcus sp.

1164


vanC112
Vancomycin
Enterococcus gallinarum

1174






1058-1059


vanC212
Vancomycin
Enterococcus casseliflavus
U94521
1060-1063





U94522





U94523





U94524





U94525





L29638


vanC312
Vancomycin
Enterococcus flavescens
L29639
1064-1066





U72706


vanD18
Vancomycin
Enterococcus faecium
AF130997


vanE12
Vancomycin
Enterococcus faecium
AF136925


tetB19
Tetracycline
Gram-negative bacteria
J01830





AF162223





AP000342





S83213





U81141





V00611


tetM19
Tetracycline
Gram-negative and
X52632




Gram-positive bacteria
AF116348





U50983





X92947





M211136





U08812





X04388


sul II20
Sulfonamides
Gram-negative bacteria
M36657





AF017389





AF017391


dhfrIa20
Trimethoprim
Gram-negative bacteria
AJ238350





X17477





K00052





U09476





X00926


dhfrIb20
Trimethoprim
Gram-negative bacteria
Z50805





Z50804


dhfrV20
Trimethoprim
Gram-negative bacteria
X12868


dhfrVI20
Trimethoprim
Gram-negative bacteria
Z86002


dhfrVII20
Trimethoprim
Gram-negative bacteria
U31119





AF139109





X58425


dhfrVIII20
Trimethoprim
Gram-negative bacteria
U10186





U09273


dhfrIX20
Trimethoprim
Gram-negative bacteria
X57730


dhfrXII20
Trimethoprim
Gram-negative bacteria
Z21672





AF175203





AF180731





M84522


dhfrXIII20
Trimethoprim
Gram-negative bacteria
Z50802


dhfrXV20
Trimethoprim
Gram-negative bacteria
Z83331


dhfrXVII20
Trimethoprim
Gram-negative bacteria
AF170088





AF180469





AF169041


dfrA20
Trimethoprim
Staphylococcus sp.
AF045472





U40259





AF051916





X13290





Y07536





Z16422





Z48233





1Bacteria having high incidence for the specified antibiotic resistance gene. The presence of the antibiotic resistance genes in other bacteria is not excluded.


2Shaw, K. J., P. N. Rather, R. S. Hare, and G. H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbial. Rev. 57: 138-163.


3Antibiotic resistance genes from our assigned U.S. Pat. No. 6,001,564 for which we have selected PCR primer pairs.


4These SEQ ID NOs. refer to a previous patent (publication WO98/20157).


5Bush, K., G. A. Jacoby and A. Medeiros. 1995. A functional classification scheme for β-lactamase and its correlation with molecular structure. Antimicrob. Agents. Chemother. 39: 1211-1233.


6Nucleotide mutations in blaSHV, blaTEM, and blaOXA, are associated with extended-spectrum β-lactamase or inhibitor-resistant β-lactamase.


7Bauerfeind, A., Y. Chong, and K. Lee. 1998. Plasmid-encoded AmpC beta-lactamases: how far have we gone 10 ears after discovery? Yonsei Med. J. 39: 520-525.


8Sutcliffe. J., T. Grebe, A. Tait-Kamradt, and L. Wondrack. 1996. Detection of erythromycin-resistant determinants by PCR. Antimicrob. Agent Chemother. 40: 2562-2566.


9Leclerc, R., A., Brisson-Noël, J. Duval, and P. Courvalin. 1991. Phenotypic expression and genetic heterogeneity of lincosamide inactivation in Staphylococcus sp. Antimicrob. Agents. Chemother. 31: 1887-1891.


10Bozdogan, B., L. Berrezouga, M.-S. Kuo, D. A. Yurek, K. A. Farley, B. J. Stockman, and R. Leclercq. 1999. A new gene, linB, conferring resistance to lincosamides by nucleotidylation in Enterococcus faecium HM1025. Antimicrob. Agents. Chemother. 43: 925-929.


11Cockerill III, F. R. 1999. Genetic methods for assessing antimicrobial resistance. Antimicrob. Agents. Chemother. 43: 199-212.


12Tenover, F. C., T. Popovic, and O Olsvik. 1996. Genetic methods for detecting antibacterial resistance genes. pp. 1368-1378. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, R. H. Yolken (eds). Manual of clinical microbiology. 6th ed., ASM Press, Washington, D. C. USA


13Dowson, C. G., T. J. Tracey, and B. G. Spratt. 1994. Origin and molecular epidemiology of penicillin-binding-protein-mediated resistance to β-lactam antibiotics. Trends Molec. Microbiol.2: 361-366.


14Jensen, L. B., N. Frimodt-Moller, F. M. Aarestrup. 1999. Presence of erm gene classes in Gram-positive bacteria of animal and human origin in Denmark. FEMS Microbiol. 170: 151-158:


15Thal, L. A., and M. J. Zervos. 1999. Occurrence and epidemiology of resistance to virginimycin and streptrogramins. J. Antimicrob. Chemother. 43: 171-176.


16Martinez J. L., A. Alonso, J. M. Gomez-Gomez, and F. Baquero. 1998. Quinolone resistance by mutations in chromosomal gyrase genes. Just the tip of the iceberg? J. Antimicrob. Chemother. 42: 683-688


17Cockerill III, F. R. 1999. Genetic methods for assessing antimicrobial resistance. Antimicrob. Agents. Chemother. 43: 199-212.


18Casadewall, B. and P. Courvalin. 1999 Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM 4339. J. Bacteriol. 181: 3644-3648.


19Roberts, M. C. 1999. Genetic mobility and distribution of tetracycline resistance determinants. Ciba Found. Symp. 207: 206-222.


20Huovinen, P., L. Sundstrom, G. Swedberg, and O. Skold. 1995. Trimethoprim and sulfonamide resistance. Antimicrob. Agent Chemother. 39: 279-289.













TABLE 6







List of bacterial toxins selected for diagnostic purposes.









Organism
Toxin
Accession number





Actinobacillus actinomycetemcomitans
Cytolethal distending toxin (cdtA, cdtB, cdtC)
AF006830



Leukotoxin (ltxA)
M27399


Actinomyces pyogenes
Hemolysin (pyolysin)
U84782


Aeromonas hydrophila
Aerolysin (aerA)
M16495



Haemolysin (hlyA)
U81555



Cytotonic enterotoxin (alt)
L77573


Bacillus anthracis
Anthrax toxin (cya)
M23179


Bacillus cereus
Enterotoxin (bceT)
D17312




AF192766, AF192767



Enterotoxic hemolysin BL
AJ237785



Non-haemolytic enterotoxins A, B and C (nhe)
Y19005


Bacillus mycoides
Hemolytic enterotoxin HBL
AJ243150 to AJ243153


Bacillus pseudomycoides
Hemolytic enterotoxin HBL
AJ243154 to AJ243156


Bacteroides fragilis
Enterotoxin (bftP)
U67735



Matrix metalloprotease/enterotoxin (fragilysin)
S75941, AF038459



Metalloprotease toxin-2
U90931




AF081785



Metalloprotease toxin-3
AF056297


Bordetella bronchiseptica
Adenylate cyclase hemolysin (cyaA)
Z37112, U22953



Dermonecrotic toxin (dnt)
U59687




AB020025


Bordetella pertussis
Pertussis toxin (S1 subunit, tox)
AJ006151




AJ006153




AJ006155




AJ006157




AJ006159




AJ007363




M14378, M16494




AJ007364




M13223




X16347



Adenyl cyclase (cya)
18323



Dermonecrotic toxin (dnt)
U10527


Campylobacter jejuni
Cytolethal distending toxin (cdtA, cdtB, cdtC)
U51121


Citrobacter freundii
Shiga-like toxin (slt-llcA)
X67514, S53206


Clostridium botulinum
Botulism toxin (BoNT) (A, B, E and F serotypes
X52066, X52088



are neurotoxic for humans; the other serotypes
X73423



have not been considered)
M30196




X70814




X70819




X71343




Z11934




X70817




M81186




X70818




X70815




X62089




X62683




S76749




X81714




X70816




X70820




X70281




L35496




M92906


Clostridium difficile
A toxin (enterotoxin) (tcdA) (cdtA)
AB012304




AF053400




Y12616




X51797




X17194




M30307



B toxin (cytotoxin) (toxB) (cdtB)
Z23277




X53138


Clostridium perfringens
Alpha (phospholipase C) (cpa)
L43545




L43546




L43547




L43548




X13608




X17300




D10248



Beta (dermonecrotic protein) (cpb)
L13198




X83275




L77965



Enterotoxin (cpe)
AJ000766




M98037




X81849




X71844




Y16009



Enterotoxin pseudogene (not expressed)
AF037328




AF037329




AF037330



Epsilon toxin (etxD)
M80837




M95206




X60694



Iota (Ia and Ib)
X73562



Lambda (metalloprotease)
D45904



Theta (perfringolysin O)
M36704


Clostridium sordellii
Cytotoxin L
X82638


Clostridium tetani
Tetanos toxin
X06214




X04436


Corynebacterium diphtheriae
Diphtheriae toxin
X00703


Corynebacterium pseudotuberculosis
Phospholipase C
A21336


Eikenella corrodens
lysine decarboxylase (cadA)
U89166


Enterobacter cloacae
Shiga-like toxin II
Z50754, U33502


Enterococcus faecalis
Cytolysin B (cylB)
M38052


Escherichia coli (EHEC)
Hemolysin toxin (hlyA and ehxA)
AF043471




X94129




X79839




X86087




AB011549




AF074613
















TABLE 7







Origin of the nucleic acids and/or sequences in the sequence listing.










SEQ ID NO.
Archaeal, bacterial, fungal or parasitical species
Source
Gene*













1
Acinetobacter baumannii
This patent
tuf


2
Actinomyces meyeri
This patent
tuf


3
Aerococcus viridans
This patent
tuf


4
Achromobacter xylosoxidans subsp. denitrificans
This patent
tuf


5
Anaerorhabdus furcosus
This patent
tuf


6
Bacillus anthracis
This patent
tuf


7
Bacillus cereus
This patent
tuf


8
Bacteroides distasonis
This patent
tuf


9
Enterococcus casseliflavus
This patent
tuf


10
Staphylococcus saprophyticus
This patent
tuf


11
Bacteroides ovatus
This patent
tuf


12
Bartonella henselae
This patent
tuf


13
Bifidobacterium adolescentis
This patent
tuf


14
Bifidobacterium dentium
This patent
tuf


15
Brucella abortus
This patent
tuf


16
Burkholderia cepacia
This patent
tuf


17
Cedecea davisae
This patent
tuf


18
Cedecea neteri
This patent
tuf


19
Cedecea lapagei
This patent
tuf


20
Chlamydia pneumoniae
This patent
tuf


21
Chlamydia psittaci
This patent
tuf


22
Chlamydia trachomatis
This patent
tuf


23
Chryseobacterium meningosepticum
This patent
tuf


24
Citrobacter amalonaticus
This patent
tuf


25
Citrobacter braakii
This patent
tuf


26
Citrobacter koseri
This patent
tuf


27
Citrobacter farmeri
This patent
tuf


28
Citrobacter freundii
This patent
tuf


29
Citrobacter sedlakii
This patent
tuf


30
Citrobacter werkmanii
This patent
tuf


31
Citrobacter youngae
This patent
tuf


32
Clostridium perfringens
This patent
tuf


33
Comamonas acidovorans
This patent
tuf


34
Corynebacterium bovis
This patent
tuf


35
Corynebacterium cervicis
This patent
tuf


36
Corynebacterium flavescens
This patent
tuf


37
Corynebacterium kutscheri
This patent
tuf


38
Corynebacterium minutissimum
This patent
tuf


39
Corynebacterium mycetoides
This patent
tuf


40
Corynebacterium pseudogenitalium
This patent
tuf


41
Corynebacterium renale
This patent
tuf


42
Corynebacterium ulcerans
This patent
tuf


43
Corynebacterium urealyticum
This patent
tuf


44
Corynebacterium xerosis
This patent
tuf


45
Coxiella burnetii
This patent
tuf


46
Edwardsiella hoshinae
This patent
tuf


47
Edwardsiella tarda
This patent
tuf


48
Eikenella corrodens
This patent
tuf


49
Enterobacter aerogenes
This patent
tuf


50
Enterobacter agglomerans
This patent
tuf


51
Enterobacter amnigenus
This patent
tuf


52
Enterobacter asburiae
This patent
tuf


53
Enterobacter cancerogenus
This patent
tuf


54
Enterobacter cloacae
This patent
tuf


55
Enterobacter gergoviae
This patent
tuf


56
Enterobacter hormaechei
This patent
tuf


57
Enterobacter sakazakii
This patent
tuf


58
Enterococcus casseliflavus
This patent
tuf


59
Enterococcus cecorum
This patent
tuf


60
Enterococcus dispar
This patent
tuf


61
Enterococcus durans
This patent
tuf


62
Enterococcus faecalis
This patent
tuf


63
Enterococcus faecalis
This patent
tuf


64
Enterococcus faecium
This patent
tuf


65
Enterococcus flavescens
This patent
tuf


66
Enterococcus gallinarum
This patent
tuf


67
Enterococcus hirae
This patent
tuf


68
Enterococcus mundtii
This patent
tuf


69
Enterococcus pseudoavium
This patent
tuf


70
Enterococcus raffinosus
This patent
tuf


71
Enterococcus saccharolyticus
This patent
tuf


72
Enterococcus solitarius
This patent
tuf


73
Enterococcus casseliflavus
This patent
tuf (C)


74
Staphylococcus saprophyticus
This patent
unknown


75
Enterococcus flavescens
This patent
tuf (C)


76
Enterococcus gallinarum
This patent
tuf (C)


77
Ehrlichia canis
This patent
tuf


78
Escherichia coli
This patent
tuf


79
Escherichia fergusonii
This patent
tuf


80
Escherichia hermannii
This patent
tuf


81
Escherichia vulneris
This patent
tuf


82
Eubacterium lentum
This patent
tuf


83
Eubacterium nodatum
This patent
tuf


84
Ewingella americana
This patent
tuf


85
Francisella tularensis
This patent
tuf


86
Fusobacterium nucleatum subsp. polymorphum
This patent
tuf


87
Gemella haemolysans
This patent
tuf


88
Gemella morbillorum
This patent
tuf


89
Haemophilus actinomycetemcomitans
This patent
tuf


90
Haemophilus aphrophilus
This patent
tuf


91
Haemophilus ducreyi
This patent
tuf


92
Haemophilus haemolyticus
This patent
tuf


93
Haemophilus parahaemolyticus
This patent
tuf


94
Haemophilus parainfluenzae
This patent
tuf


95
Haemophilus paraphrophilus
This patent
tuf


96
Haemophilus segnis
This patent
tuf


97
Hafnia alvei
This patent
tuf


98
Kingella kingae
This patent
tuf


99
Klebsiella omithinolytica
This patent
tuf


100
Klebsiella oxytoca
This patent
tuf


101
Klebsiella planticola
This patent
tuf


102
Klebsiella pneumoniae subsp. ozaenae
This patent
tuf


103
Klebsiella pneumoniae pneumoniae
This patent
tuf


104
Klebsiella pneumoniae subsp. rhinoscleromatis
This patent
tuf


105
Kluyvera ascorbata
This patent
tuf


106
Kluyvera cryocrescens
This patent
tuf


107
Kluyvera georgiana
This patent
tuf


108
Lactobacillus casei subsp. casei
This patent
tuf


109
Lactococcus lactis subsp. lactis
This patent
tuf


110
Leclercia adecarboxylata
This patent
tuf


111
Legionella micdadei
This patent
tuf


112
Legionella pneumophila subsp. pneumophila
This patent
tuf


113
Leminorella grimontii
This patent
tuf


114
Leminorella richardii
This patent
tuf


115
Leptospira interrogans
This patent
tuf


116
Megamonas hypermegale
This patent
tuf


117
Mitsuokella multacidus
This patent
tuf


118
Mobiluncus curtisii subsp. holmesii
This patent
tuf


119
Moellerella wisconsensis
This patent
tuf


120
Moraxella catarrhalis
This patent
tuf


121
Morganella morganii subsp. morganii
This patent
tuf


122
Mycobacterium tuberculosis
This patent
tuf


123
Neisseria cinerea
This patent
tuf


124
Neisseria elongata subsp. elongata
This patent
tuf


125
Neisseria flavescens
This patent
tuf


126
Neisseria gonorrhoeae
This patent
tuf


127
Neisseria lactamica
This patent
tuf


128
Neisseria meningitidis
This patent
tuf


129
Neisseria mucosa
This patent
tuf


130
Neisseria sicca
This patent
tuf


131
Neisseria subflava
This patent
tuf


132
Neisseria weaveri
This patent
tuf


133
Ochrobactrum anthropi
This patent
tuf


134
Pantoea agglomerans
This patent
tuf


135
Pantoea dispersa
This patent
tuf


136
Pasteurella multocida
This patent
tuf


137
Peptostreptococcus anaerobius
This patent
tuf


138
Peptostreptococcus asaccharolyticus
This patent
tuf


139
Peptostreptococcus prevotii
This patent
tuf


140
Porphyromonas asaccharolytica
This patent
tuf


141
Porphyromonas gingivalis
This patent
tuf


142
Pragia fontium
This patent
tuf


143
Prevotella melaninogenica
This patent
tuf


144
Prevotella oralis
This patent
tuf


145
Propionibacterium acnes
This patent
tuf


146
Proteus mirabilis
This patent
tuf


147
Proteus penneri
This patent
tuf


148
Proteus vulgaris
This patent
tuf


149
Providencia alcalifaciens
This patent
tuf


150
Providencia rettgeri
This patent
tuf


151
Providencia rustigianii
This patent
tuf


152
Providencia stuartii
This patent
tuf


153
Pseudomonas aeruginosa
This patent
tuf


154
Pseudomonas fluorescens
This patent
tuf


155
Pseudomonas stutzeri
This patent
tuf


156
Psychrobacter phenylpyruvicum
This patent
tuf


157
Rahnella aquatilis
This patent
tuf


158
Salmonella choleraesuis subsp. arizonae
This patent
tuf


159
Salmonella choleraesuis subsp. choleraesuis
This patent
tuf



serotype Choleraesuis


160
Salmonella choleraesuis subsp. diarizonae
This patent
tuf


161
Salmonella choleraesuis subsp. choleraesuis
This patent
tuf



serotype Heidelberg


162
Salmonella choleraesuis subsp. houtenae
This patent
tuf


163
Salmonella choleraesuis subsp. indica
This patent
tuf


164
Salmonella choleraesuis subsp. salamae
This patent
tuf


165
Salmonella choleraesuis subsp. choleraesuis serotype Typhi
This patent
tuf


166
Serratia fonticola
This patent
tuf


167
Serratia liquefaciens
This patent
tuf


168
Serratia marcescens
This patent
tuf


169
Serratia odorifera
This patent
tuf


170
Serratia plymuthica
This patent
tuf


171
Serratia rubidaea
This patent
tuf


172
Shigella boydii
This patent
tuf


173
Shigella dysenteriae
This patent
tuf


174
Shigella flexneri
This patent
tuf


175
Shigella sonnei
This patent
tuf


176
Staphylococcus aureus
This patent
tuf


177
Staphylococcus aureus
This patent
tuf


178
Staphylococcus aureus
This patent
tuf


179
Staphylococcus aureus
This patent
tuf


180
Staphylococcus aureus subsp. aureus
This patent
tuf


181
Staphylococcus auricularis
This patent
tuf


182
Staphylococcus capitis subsp. capitis
This patent
tuf


183
Macrococcus caseolyticus
This patent
tuf


184
Staphylococcus cohnii subsp. cohnii
This patent
tuf


185
Staphylococcus epidermidis
This patent
tuf


186
Staphylococcus haemolyticus
This patent
tuf


187
Staphylococcus warneri
This patent
tuf


188
Staphylococcus haemolyticus
This patent
tuf


189
Staphylococcus haemolyticus
This patent
tuf


190
Staphylococcus haemolyticus
This patent
tuf


191
Staphylococcus hominis subsp. hominis
This patent
tuf


192
Staphylococcus warneri
This patent
tuf


193
Staphylococcus hominis
This patent
tuf


194
Staphylococcus hominis
This patent
tuf


195
Staphylococcus hominis
This patent
tuf


196
Staphylococcus hominis
This patent
tuf


197
Staphylococcus lugdunensis
This patent
tuf


198
Staphylococcus saprophyticus
This patent
tuf


199
Staphylococcus saprophyticus
This patent
tuf


200
Staphylococcus saprophyticus
This patent
tuf


201
Staphylococcus sciuri subsp. sciuri
This patent
tuf


202
Staphylococcus warneri
This patent
tuf


203
Staphylococcus warneri
This patent
tuf


204
Bifidobacterium longum
This patent
tuf


205
Stenotrophomonas maltophilia
This patent
tuf


206
Streptococcus acidominimus
This patent
tuf


207
Streptococcus agalactiae
This patent
tuf


208
Streptococcus agalactiae
This patent
tuf


209
Streptococcus agalactiae
This patent
tuf


210
Streptococcus agalactiae
This patent
tuf


211
Streptococcus anginosus
This patent
tuf


212
Streptococcus bovis
This patent
tuf


213
Streptococcus anginosus
This patent
tuf


214
Streptococcus cricetus
This patent
tuf


215
Streptococcus cristatus
This patent
tuf


216
Streptococcus downei
This patent
tuf


217
Streptococcus dysgalactiae
This patent
tuf


218
Streptococcus equi subsp. equi
This patent
tuf


219
Streptococcus ferus
This patent
tuf


220
Streptococcus gordonii
This patent
tuf


221
Streptococcus anginosus
This patent
tuf


222
Streptococcus macacae
This patent
tuf


223
Streptococcus gordonii
This patent
tuf


224
Streptococcus mutans
This patent
tuf


225
Streptococcus parasanguinis
This patent
tuf


226
Streptococcus ratti
This patent
tuf


227
Streptococcus sanguinis
This patent
tuf


228
Streptococcus sobrinus
This patent
tuf


229
Streptococcus suis
This patent
tuf


230
Streptococcus uberis
This patent
tuf


231
Streptococcus vestibularis
This patent
tuf


232
Tatumella ptyseos
This patent
tuf


233
Trabulsiella guamensis
This patent
tuf


234
Veillonella parvula
This patent
tuf


235
Yersinia enterocolitica
This patent
tuf


236
Yersinia frederiksenii
This patent
tuf


237
Yersinia intermedia
This patent
tuf


238
Yersinia pestis
This patent
tuf


239
Yersinia pseudotuberculosis
This patent
tuf


240
Yersinia rohdei
This patent
tuf


241
Yokenella regensburgei
This patent
tuf


242
Achromobacter xylosoxidans subsp. denitrificans
This patent
atpD


243
Acinetobacter baumannii
This patent
atpD


244
Acinetobacter lwoffii
This patent
atpD


245
Staphylococcus saprophyticus
This patent
atpD


246
Alcaligenes faecalis subsp. faecalis
This patent
atpD


247
Bacillus anthracis
This patent
atpD


248
Bacillus cereus
This patent
atpD


249
Bacteroides distasonis
This patent
atpD


250
Bacteroides ovatus
This patent
atpD


251
Leclercia adecarboxylata
This patent
atpD


252
Stenotrophomonas maltophilia
This patent
atpD


253
Bartonella henselae
This patent
atpD


254
Bifidobacterium adolescentis
This patent
atpD


255
Brucella abortus
This patent
atpD


256
Cedecea davisae
This patent
atpD


257
Cedecea lapagei
This patent
atpD


258
Cedecea neteri
This patent
atpD


259
Chryseobacterium meningosepticum
This patent
atpD


260
Citrobacter amalonaticus
This patent
atpD


261
Citrobacter braakii
This patent
atpD


262
Citrobacter koseri
This patent
atpD


263
Citrobacter farmeri
This patent
atpD


264
Citrobacter freundii
This patent
atpD


265
Citrobacter koseri
This patent
atpD


266
Citrobacter sedlakii
This patent
atpD


267
Citrobacter werkmanii
This patent
atpD


268
Citrobacter youngae
This patent
atpD


269
Clostridium innocuum
This patent
atpD


270
Clostridium perfringens
This patent
atpD


272
Corynebacterium diphtheriae
This patent
atpD


273
Corynebacterium pseudodiphtheriticum
This patent
atpD


274
Corynebacterium ulcerans
This patent
atpD


275
Corynebacterium urealyticum
This patent
atpD


276
Coxiella burnetii
This patent
atpD


277
Edwardsiella hoshinae
This patent
atpD


278
Edwardsiella tarda
This patent
atpD


279
Eikenella corrodens
This patent
atpD


280
Enterobacter agglomerans
This patent
atpD


281
Enterobacter arnnigenus
This patent
atpD


282
Enterobacter asburiae
This patent
atpD


283
Enterobacter cancerogenus
This patent
atpD


284
Enterobacter cloacae
This patent
atpD


285
Enterobacter gergoviae
This patent
atpD


286
Enterobacter hormaechei
This patent
atpD


287
Enterobacter sakazakii
This patent
atpD


288
Enterococcus avium
This patent
atpD


289
Enterococcus casseliflavus
This patent
atpD


290
Enterococcus durans
This patent
atpD


291
Enterococcus faecalis
This patent
atpD


292
Enterococcus faecium
This patent
atpD


293
Enterococcus gallinarum
This patent
atpD


294
Enterococcus saccharolyticus
This patent
atpD


295
Escherichia fergusonii
This patent
atpD


296
Escherichia hermannii
This patent
atpD


297
Escherichia vulneris
This patent
atpD


298
Eubacterium lentum
This patent
atpD


299
Ewingella americana
This patent
atpD


300
Francisella tularensis
This patent
atpD


301
Fusobacterium gonidiaformans
This patent
atpD


302
Fusobacterium necrophorum subsp. necrophorum
This patent
atpD


303
Fusobacterium nucleatum subsp. polymorphum
This patent
atpD


304
Gardnerella vaginalis
This patent
atpD


305
Gemella haemolysans
This patent
atpD


306
Gemella morbillorum
This patent
atpD


307
Haemophilus ducreyi
This patent
atpD


308
Haemophilus haemolyticus
This patent
atpD


309
Haemophilus parahaemolyticus
This patent
atpD


310
Haemophilus parainfluenzae
This patent
atpD


311
Hafnia alvei
This patent
atpD


312
Kingella kingae
This patent
atpD


313
Klebsiella pneumoniae subsp. ozaenae
This patent
atpD


314
Klebsiella omithinolytica
This patent
atpD


315
Klebsiella oxytoca
This patent
atpD


316
Klebsiella planticola
This patent
atpD


317
Kiebsiella pneumoniae subsp. pneumoniae
This patent
atpD


318
Kluyvera ascorbata
This patent
atpD


319
Kluyvera cryocrescens
This patent
atpD


320
Kluyvera georgiana
This patent
atpD


321
Lactobacillus acidophilus
This patent
atpD


322
Legionella pneumophila subsp. pneumophila
This patent
atpD


323
Leminorella grimontii
This patent
atpD


324
Listeria monocytogenes
This patent
atpD


325
Micrococcus lylae
This patent
atpD


326
Moellerella wisconsensis
This patent
atpD


327
Moraxella catarrhalis
This patent
atpD


328
Moraxella osloensis
This patent
atpD


329
Morganella morganii subsp. morganii
This patent
atpD


330
Pantoea agglomerans
This patent
atpD


331
Pantoea dispersa
This patent
atpD


332
Pasteurella multocida
This patent
atpD


333
Pragia fontium
This patent
atpD


334
Proteus mirabilis
This patent
atpD


335
Proteus vulgaris
This patent
atpD


336
Providencia alcalifaciens
This patent
atpD


337
Providencia rettgeri
This patent
atpD


338
Providencia rustigianii
This patent
atpD


339
Providencia stuartii
This patent
atpD


340
Psychrobacter phenylpyruvicum
This patent
atpD


341
Rahnella aquatilis
This patent
atpD


342
Salmonella choleraesuis subsp. arizonae
This patent
atpD


343
Salmonella choleraesuis subsp. choleraesuis
This patent
atpD



serotype Choleraesuis


344
Salmonella choleraesuis subsp. diarizonae
This patent
atpD


345
Salmonella choleraesuis subsp. houtenae
This patent
atpD


346
Salmonella choleraesuis subsp. indica
This patent
atpD


347
Salmonella choleraesuis subsp. choleraesuis
This patent
atpD



serotype Paratyphi A


348
Salmonella choleraesuis subsp. choleraesuis
This patent
atpD



serotype Paratyphi B


349
Salmonella choleraesuis subsp. salamae
This patent
atpD


350
Salmonella choleraesuis subsp. choleraesuis
This patent
atpD



serotype Typhi


351
Salmonella choleraesuis subsp. choleraesuis
This patent
atpD



serotype Typhimurium


352
Salmonella choleraesuis subsp. choleraesuis
This patent
atpD



serotype Virchow


353
Serratia ficaria
This patent
atpD


354
Serratia fonticola
This patent
atpD


355
Serratia grimesii
This patent
atpD


356
Serratia liquefaciens
This patent
atpD


357
Serratia marcescens
This patent
atpD


358
Serratia odorifera
This patent
atpD


359
Serratia plymuthica
This patent
atpD


360
Serratia rubidaea
This patent
atpD


361
Pseudomonas putida
This patent
atpD


362
Shigella boydii
This patent
atpD


363
Shigella dysenteriae
This patent
atpD


364
Shigella flexneri
This patent
atpD


365
Shigella sonnei
This patent
atpD


366
Staphylococcus aureus
This patent
atpD


367
Staphylococcus auricularis
This patent
atpD


368
Staphylococcus capitis subsp. capitis
This patent
atpD


369
Staphylococcus cohnii subsp. cohnii
This patent
atpD


370
Staphylococcus epidermidis
This patent
atpD


371
Staphylococcus haemolyticus
This patent
atpD


372
Staphylococcus hominis subsp. hominis
This patent
atpD


373
Staphylococcus hominis
This patent
atpD


374
Staphylococcus lugdunensis
This patent
atpD


375
Staphylococcus saprophyticus
This patent
atpD


376
Staphylococcus simulans
This patent
atpD


377
Staphylococcus wameri
This patent
atpD


378
Streptococcus acidominimus
This patent
atpD


379
Streptococcus agalactiae
This patent
atpD


380
Streptococcus agalactiae
This patent
atpD


381
Streptococcus agalactiae
This patent
atpD


382
Streptococcus agalactiae
This patent
atpD


383
Streptococcus agalactiae
This patent
atpD


384
Streptococcus dysgalactiae
This patent
atpD


385
Streptococcus equi subsp. equi
This patent
atpD


386
Streptococcus anginosus
This patent
atpD


387
Streptococcus salivarius
This patent
atpD


388
Streptococcus suis
This patent
atpD


389
Streptococcus uberis
This patent
atpD


390
Tatumella ptyseos
This patent
atpD


391
Trabulsiella guamensis
This patent
atpD


392
Yersinia bercovieri
This patent
atpD


393
Yersinia enterocolitica
This patent
atpD


394
Yersinia frederiksenii
This patent
atpD


395
Yersinia intermedia
This patent
atpD


396
Yersinia pseudotuberculosis
This patent
atpD


397
Yersinia rohdei
This patent
atpD


398
Yokenella regensburgei
This patent
atpD


399
Yarrowia lipolytica
This patent
tuf (EF-1)


400
Absidia corymbifera
This patent
tuf (EF-1)


401
Alternaria alternata
This patent
tuf (EF-1)


402
Aspergillus flavus
This patent
tuf (EF-1)


403
Aspergillus fumigatus
This patent
tuf (EF-1)


404
Aspergillus fumigatus
This patent
tuf (EF-1)


405
Aspergillus niger
This patent
tuf (EF-1)


406
Blastoschizomyces capitatus
This patent
tuf (EF-1)


407
Candida albicans
This patent
tuf (EF-1)


408
Candida albicans
This patent
tuf (EF-1)


409
Candida albicans
This patent
tuf (EF-1)


410
Candida albicans
This patent
tuf (EF-1)


411
Candida albicans
This patent
tuf (EF-1)


412
Candida dubliniensis
This patent
tuf (EF-1)


413
Candida catenulata
This patent
tuf (EF-1)


414
Candida dubliniensis
This patent
tuf (EF-1)


415
Candida dubliniensis
This patent
tuf (EF-1)


416
Candida famata
This patent
tuf (EF-1)


417
Candida glabrata
WO98/20157
tuf (EF-1)


418
Candida guilliermondii
This patent
tuf (EF-1)


419
Candida haemulonii
This patent
tuf (EF-1)


420
Candida inconspicua
This patent
tuf (EF-1)


421
Candida kefyr
This patent
tuf (EF-1)


422
Candida krusei
WO98/20157
tuf (EF-1)


423
Candida lambica
This patent
tuf (EF-1)


424
Candida lusitaniae
This patent
tuf (EF-1)


425
Candida norvegensis
This patent
tuf (EF-1)


426
Candida parapsilosis
WO98/20157
tuf (EF-1)


427
Candida rugosa
This patent
tuf (EF-1)


428
Candida sphaerica
This patent
tuf (EF-1)


429
Candida tropicalis
WO98/20157
tuf (EF-1)


430
Candida utilis
This patent
tuf (EF-1)


431
Candida viswanathii
This patent
tuf (EF-1)


432
Candida zeylanoides
This patent
tuf (EF-1)


433
Coccidioides immitis
This patent
tuf (EF-1)


434
Cryptococcus albidus
This patent
tuf (EF-1)


435
Exophiala jeanselmei
This patent
tuf (EF-1)


436
Fusarium oxysporum
This patent
tuf (EF-1)


437
Geotrichum sp.
This patent
tuf (EF-1)


438
Histoplasma capsulatum
This patent
tuf (EF-1)


439
Issatchenkia orientalis Kudrjanzev
This patent
tuf (EF-1)


440
Malassezia furfur
This patent
tuf (EF-1)


441
Malassezia pachydermatis
This patent
tuf (EF-1)


442
Malbranchea filamentosa
This patent
tuf (EF-1)


443
Metschnikowia pulcherrima
This patent
tuf (EF-1)


444
Paecilomyces lilacinus
This patent
tuf (EF-1)


445
Paracoccidioides brasiliensis
This patent
tuf (EF-1)


446
Penicillium marneffei
This patent
tuf (EF-1)


447
Pichia anomala
This patent
tuf (EF-1)


448
Pichia anomala
This patent
tuf (EF-1)


449
Pseudallescheria boydii
This patent
tuf (EF-1)


450
Rhizopus oryzae
This patent
tuf (EF-1)


451
Rhodotorula minuta
This patent
tuf (EF-1)


452
Sporobolomyces salmonicolor
This patent
tuf (EF-1)


453
Sporothrix schenckii
This patent
tuf (EF-1)


454
Stephanoascus ciferrii
This patent
tuf (EF-1)


455
Trichophyton mentagrophytes
This patent
tuf (EF-1)


456
Trichosporon cutaneum
This patent
tuf (EF-1)


457
Wangiella dermatitidis
This patent
tuf (EF-1)


458
Aspergillus fumigatus
This patent
atpD


459
Blastoschizomyces capitatus
This patent
atpD


460
Candida albicans
This patent
atpD


461
Candida dubliniensis
This patent
atpD


462
Candida famata
This patent
atpD


463
Candida glabrata
This patent
atpD


464
Candida guilliermondii
This patent
atpD


465
Candida haemulonii
This patent
atpD


466
Candida inconspicua
This patent
atpD


467
Candida kefyr
This patent
atpD


468
Candida krusei
This patent
atpD


469
Candida lambica
This patent
atpD


470
Candida lusitaniae
This patent
atpD


471
Candida norvegensis
This patent
atpD


472
Candida parapsilosis
This patent
atpD


473
Candida rugosa
This patent
atpD


474
Candida sphaerica
This patent
atpD


475
Candida tropicalis
This patent
atpD


476
Candida utilis
This patent
atpD


477
Candida viswanathii
This patent
atpD


478
Candida zeylanoides
This patent
atpD


479
Coccidioides immitis
This patent
atpD


480
Cryptococcus albidus
This patent
atpD


481
Fusarium oxysporum
This patent
atpD


482
Geotrichum sp.
This patent
atpD


483
Histoplasma capsulatum
This patent
atpD


484
Malassezia furfur
This patent
atpD


485
Malassezia pachydermatis
This patent
atpD


486
Metschnikowia pulcherrima
This patent
atpD


487
Penicillium marneffei
This patent
atpD


488
Pichia anomala
This patent
atpD


489
Pichia anomala
This patent
atpD


490
Rhodotorula minuta
This patent
atpD


491
Rhodotorula mucilaginosa
This patent
atpD


492
Sporobolomyces salmonicolor
This patent
atpD


493
Sporothrix schenckii
This patent
atpD


494
Stephanoascus ciferrii
This patent
atpD


495
Trichophyton mentagrophytes
This patent
atpD


496
Wangiella dermatitidis
This patent
atpD


497
Yarrowia lipolytica
This patent
atpD


498
Aspergillus fumigatus
This patent
tuf (M)


499
Blastoschizomyces capitatus
This patent
tuf (M)


500
Candida rugosa
This patent
tuf (M)


501
Coccidioides immitis
This patent
tuf (M)


502
Fusarium oxysporum
This patent
tuf (M)


503
Histoplasma capsulatum
This patent
tuf (M)


504
Paracoccidioides brasiliensis
This patent
tuf (M)


505
Penicillium marneffei
This patent
tuf (M)


506
Pichia anomala
This patent
tuf (M)


507
Trichophyton mentagrophytes
This patent
tuf (M)


508
Yarrowia lipolytica
This patent
tuf (M)


509
Babesia bigemina
This patent
tuf (EF-1)


510
Babesia bovis
This patent
tuf (EF-1)


511
Crithidia fasciculata
This patent
tuf (EF-1)


512
Entamoeba histolytica
This patent
tuf (EF-1)


513
Giardia lamblia
This patent
tuf (EF-1)


514
Leishmania tropica
This patent
tuf (EF-1)


515
Leishmania aethiopica
This patent
tuf (EF-1)


516
Leishmania tropica
This patent
tuf (EF-1)


517
Leishmania donovani
This patent
tuf (EF-1)


518
Leishmania infantum
This patent
tuf (EF-1)


519
Leishmania enriettii
This patent
tuf (EF-1)


520
Leishmania gerbilli
This patent
tuf (EF-1)


521
Leishmania hertigi
This patent
tuf (EF-1)


522
Leishmania major
This patent
tuf (EF-1)


523
Leishmania amazonensis
This patent
tuf (EF-1)


524
Leishmania mexicana
This patent
tuf (EF-1)


525
Leishmania tarentolae
This patent
tuf (EF-1)


526
Leishmania tropica
This patent
tuf (EF-1)


527
Neospora caninum
This patent
tuf (EF-1)


528
Trichomonas vaginalis
This patent
tuf (EF-1)


529
Trypanosoma brucei subsp. brucei
This patent
tuf (EF-1)


530
Crithidia fasciculata
This patent
atpD


531
Leishmania tropica
This patent
atpD


532
Leishmania aethiopica
This patent
atpD


533
Leishmania donovani
This patent
atpD


534
Leishmania infantum
This patent
atpD


535
Leishmania gerbilli
This patent
atpD


536
Leishmania hertigi
This patent
atpD


537
Leishmania major
This patent
atpD


538
Leishmania amazonensis
This patent
atpD


607
Enterococcus faecalis
WO98/20157
tuf


608
Enterococcus faecium
WO98/20157
tuf


609
Enterococcus gallinarum
WO98/20157
tuf


610
Haemophilus influenzae
WO98/20157
tuf


611
Staphylococcus epidermidis
WO98/20157
tuf


612
Salmonella choleraesuis subsp. choleraesuis
This patent
tuf



serotype Paratyphi A


613
Serratia ficaria
This patent
tuf


614
Enterococcus malodoratus
This patent
tuf (C)


615
Enterococcus durans
This patent
tuf (C)


616
Enterococcus pseudoavium
This patent
tuf (C)


617
Enterococcus dispar
This patent
tuf (C)


618
Enterococcus avium
This patent
tuf (C)


619
Saccharomyces cerevisiae
Database
tuf (M)


621
Enterococcus faecium
This patent
tuf (C)


622
Saccharomyces cerevisiae
This patent
tuf (EF-1)


623
Cryptococcus neoformans
This patent
tuf (EF-1)


624
Candida albicans
WO98/20157
tuf (EF-1)


662
Corynebacterium diphtheriae
WO98/20157
tuf


663
Candida catenulata
This patent
atpD


665
Saccharomyces cerevisiae
Database
tuf (EF-1)


666
Saccharomyces cerevisiae
Database
atpD


667
Trypanosoma cruzi
This patent
atpD


668
Corynebacterium glutamicum
Database
tuf


669
Escherichia coli
Database
atpD


670
Helicobacter pylori
Database
atpD


671
Clostridium acetobutylicum
Database
atpD


672
Cytophaga lytica
Database
atpD


673
Ehrlichia risticii
This patent
atpD


674
Vibrio cholerae
This patent
atpD


675
Vibrio cholerae
This patent
tuf


676
Leishmania enriettii
This patent
atpD


677
Babesia microti
This patent
tuf (EF-1)


678
Cryptococcus neoformans
This patent
atpD


679
Cryptococcus neoformans
This patent
atpD


680
Cunninghamella bertholletiae
This patent
atpD


684
Candida tropicalis
Database
atpD (V)


685
Enterococcus hirae
Database
atpD (V)


686
Chlamydia pneumoniae
Database
atpD (V)


687
Halobacterium salinarum
Database
atpD (V)


688
Homo sapiens
Database
atpD (V)


689
Plasmodium falciparum
Database
atpD (V)


690
Saccharomyces cerevisiae
Database
atpD (V)


691
Schizosaccharomyces pombe
Database
atpD (V)


692
Trypanosoma congolense
Database
atpD (V)


693
Thermus thermophilus
Database
atpD (V)


698
Escherichia coli
WO98/20157
tuf


709
Borrelia burgdorferi
Database
atpD (V)


710
Treponema pallidum
Database
atpD (V)


711
Chlamydia trachomatis
Genome project
atpD (V)


712
Enterococcus faecalis
Genome project
atpD (V)


713
Methanosarcina barkeri
Database
atpD (V)


714
Methanococcus jannaschii
Database
atpD (V)


715
Porphyromonas gingivalis
Genome project
atpD (V)


716
Streptococcus pneumoniae
Genome project
atpD (V)


717
Burkholderia mallei
This patent
tuf


718
Burkholderia pseudomallei
This patent
tuf


719
Clostridium beijerinckii
This patent
tuf


720
Clostridium innocuum
This patent
tuf


721
Clostridium novyi
This patent
tuf


722
Clostridium septicum
This patent
tuf


723
Clostridium tertium
This patent
tuf


724
Clostridium tetani
This patent
tuf


725
Enterococcus malodoratus
This patent
tuf


726
Enterococcus sulfureus
This patent
tuf


727
Lactococcus garvieae
This patent
tuf


728
Mycoplasma pirum
This patent
tuf


729
Mycoplasma salivarium
This patent
tuf


730
Neisseria polysaccharea
This patent
tuf


731
Salmonella choleraesuis subsp. choleraesuis
This patent
tuf



serotype Enteritidis


732
Salmonella choleraesuis subsp. choleraesuis
This patent
tuf



serotype Gallinarum


733
Salmonella choleraesuis subsp. choleraesuis
This patent
tuf



serotype Paratyphi B


734
Salmonella choleraesuis subsp. choleraesuis
This patent
tuf



serotype Virchow


735
Serratia grimesii
This patent
tuf


736
Clostridium difficile
This patent
tuf


737
Burkholderia pseudomallei
This patent
atpD


738
Clostridium bifermentans
This patent
atpD


739
Clostridium beijerinckii
This patent
atpD


740
Clostridium difficile
This patent
atpD


741
Clostridium ramosum
This patent
atpD


742
Clostridium septicum
This patent
atpD


743
Clostridium tertium
This patent
atpD


744
Comamonas acidovorans
This patent
atpD


745
Klebsiella pneumoniae subsp. rhinoscleromatis
This patent
atpD


746
Neisseria canis
This patent
atpD


747
Neisseria cinerea
This patent
atpD


748
Neisseria cuniculi
This patent
atpD


749
Neisseria elongata subsp. elongata
This patent
atpD


750
Neisseria flavescens
This patent
atpD


751
Neisseria gonorrhoeae
This patent
atpD


752
Neisseria gonorrhoeae
This patent
atpD


753
Neisseria lactamica
This patent
atpD


754
Neisseria meningitidis
This patent
atpD


755
Neisseria mucosa
This patent
atpD


756
Neisseria subflava
This patent
atpD


757
Neisseria weaveri
This patent
atpD


758
Neisseria animalis
This patent
atpD


759
Proteus penneri
This patent
atpD


760
Salmonella choleraesuis subsp. choleraesuis
This patent
atpD



serotype Enteritidis


761
Yersinia pestis
This patent
atpD


762
Burkholderia mallei
This patent
atpD


763
Clostridium sordellii
This patent
atpD


764
Clostridium novyi
This patent
atpD


765
Clostridium botulinum
This patent
atpD


766
Clostridium histolyticum
This patent
atpD


767
Peptostreptococcus prevotii
This patent
atpD


768
Absidia corymbifera
This patent
atpD


769
Alternaria alternata
This patent
atpD


770
Aspergillus flavus
This patent
atpD


771
Mucor circinelloides
This patent
atpD


772
Piedraia hortai
This patent
atpD


773
Pseudallescheria boydii
This patent
atpD


774
Rhizopus oryzae
This patent
atpD


775
Scopulariopsis koningii
This patent
atpD


776
Trichophyton mentagrophytes
This patent
atpD


777
Trichophyton tonsurans
This patent
atpD


778
Trichosporon cutaneum
This patent
atpD


779
Cladophialophora carrionii
This patent
tuf (EF-1)


780
Cunninghamella bertholletiae
This patent
tuf (EF-1)


781
Curvularia lunata
This patent
tuf (EF-1)


782
Fonsecaea pedrosoi
This patent
tuf (EF-1)


783
Microsporum audouinii
This patent
tuf (EF-1)


784
Mucor circinelloides
This patent
tuf (EF-1)


785
Phialophora verrucosa
This patent
tuf (EF-1)


786
Saksenaea vasiformis
This patent
tuf (EF-1)


787
Syncephalastrum racemosum
This patent
tuf (EF-1)


788
Trichophyton tonsurans
This patent
tuf (EF-1)


789
Trichophyton mentagrophytes
This patent
tuf (EF-1)


790
Bipolaris hawaiiensis
This patent
tuf (EF-1)


791
Aspergillus fumigatus
This patent
tuf (M)


792
Trichophyton mentagrophytes
This patent
tuf (M)


827
Clostridium novyi
This patent
atpD (V)


828
Clostridium difficile
This patent
atpD (V)


829
Clostridium septicum
This patent
atpD (V)


830
Clostridium botulinum
This patent
atpD (V)


831
Clostridium perfringens
This patent
atpD (V)


832
Clostridium tetani
This patent
atpD (V)


833
Streptococcus pyogenes
Database
atpD (V)


834
Babesia bovis
This patent
atpD (V)


835
Cryptosporidium parvum
This patent
atpD (V)


836
Leishmania infantum
This patent
atpD (V)


837
Leishmania major
This patent
atpD (V)


838
Leishmania tarentolae
This patent
atpD (V)


839
Trypanosoma brucei
This patent
atpD (V)


840
Trypanosoma cruzi
This patent
tuf (EF-1)


841
Trypanosoma cruzi
This patent
tuf (EF-1)


842
Trypanosoma cruzi
This patent
tuf (EF-1)


843
Babesia bovis
This patent
tuf (M)


844
Leishmania aethiopica
This patent
tuf (M)


845
Leishmania amazonensis
This patent
tuf (M)


846
Leishmania donovani
This patent
tuf (M)


847
Leishmania infantum
This patent
tuf (M)


848
Leishmania enriettii
This patent
tuf (M)


849
Leishmania gerbilli
This patent
tuf (M)


850
Leishmania major
This patent
tuf (M)


851
Leishmania mexicana
This patent
tuf (M)


852
Leishmania tarentolae
This patent
tuf (M)


853
Trypanosoma cruzi
This patent
tuf (M)


854
Trypanosoma cruzi
This patent
tuf (M)


855
Trypanosoma cruzi
This patent
tuf (M)


856
Babesia bigemina
This patent
atpD


857
Babesia bovis
This patent
atpD


858
Babesia microti
This patent
atpD


859
Leishmania guyanensis
This patent
atpD


860
Leishmania mexicana
This patent
atpD


861
Leishmania tropica
This patent
atpD


862
Leishmania tropica
This patent
atpD


863
Bordetella pertussis
Database
tuf


864
Trypanosoma brucei brucei
Database
tuf (EF-1)


865
Cryptosporidium parvum
This patent
tuf (EF-1)


866
Staphylococcus saprophyticus
This patent
atpD


867
Zoogloea ramigera
This patent
atpD


868
Staphylococcus saprophyticus
This patent
tuf


869
Enterococcus casseliflavus
This patent
tuf


870
Enterococcus casseliflavus
This patent
tuf


871
Enterococcus flavescens
This patent
tuf


872
Enterococcus gallinarum
This patent
tuf


873
Enterococcus gallinarum
This patent
tuf


874
Staphylococcus haemolyticus
This patent
tuf


875
Staphylococcus epidermidis
This patent
tuf


876
Staphylococcus epidermidis
This patent
tuf


877
Staphylococcus epidermidis
This patent
tuf


878
Staphylococcus epidermidis
This patent
tuf


879
Enterococcus gallinarum
This patent
tuf


880
Pseudomonas aeruginosa
This patent
tuf


881
Enterococcus casseliflavus
This patent
tuf


882
Enterococcus casseliflavus
This patent
tuf


883
Enterococcus faecalis
This patent
tuf


884
Enterococcus faecalis
This patent
tuf


885
Enterococcus faecium
This patent
tuf


886
Enterococcus faecium
This patent
tuf


887
Zoogloea ramigera
This patent
tuf


888
Enterococcus faecalis
This patent
tuf


889
Aspergillus fumigatus
This patent
atpD


890
Penicillium marneffei
This patent
atpD


891
Paecilomyces lilacinus
This patent
atpD


892
Penicillium marneffei
This patent
atpD


893
Sporothrix schenckii
This patent
atpD


894
Malbranchea filamentosa
This patent
atpD


895
Paecilomyces lilacinus
This patent
atpD


896
Aspergillus niger
This patent
atpD


897
Aspergillus fumigatus
This patent
tuf (EF-1)


898
Penicillium marneffei
This patent
tuf (EF-1)


899
Piedraia hortai
This patent
tuf (EF-1)


900
Paecilomyces lilacinus
This patent
tuf (EF-1)


901
Paracoccidioides brasiliensis
This patent
tuf (EF-1)


902
Sporothrix schenckii
This patent
tuf (EF-1)


903
Penicillium marneffei
This patent
tuf (EF-1)


904
Curvularia lunata
This patent
tuf (M)


905
Aspergillus niger
This patent
tuf (M)


906
Bipolaris hawaiiensis
This patent
tuf (M)


907
Aspergillus flavus
This patent
tuf (M)


908
Alternaria alternata
This patent
tuf (M)


909
Penicillium marneffei
This patent
tuf (M)


910
Penicillium marneffei
This patent
tuf (M)


918
Escherichia coli
Database
recA


929
Bacteroides fragilis
This patent
atpD (V)


930
Bacteroides distasonis
This patent
atpD (V)


931
Porphyromonas asaccharolytica
This patent
atpD (V)


932
Listeria monocytogenes
This patent
tuf


939
Saccharomyces cerevisiae
Database
recA (Rad51)


940
Saccharomyces cerevisiae
Database
recA (Dmc1)


941
Cryptococcus humicolus
This patent
atpD


942
Escherichia coli
This patent
atpD


943
Escherichia coli
This patent
atpD


944
Escherichia coli
This patent
atpD


945
Escherichia coli
This patent
atpD


946
Neisseria polysaccharea
This patent
atpD


947
Neisseria sicca
This patent
atpD


948
Streptococcus mitis
This patent
atpD


949
Streptococcus mitis
This patent
atpD


950
Streptococcus mitis
This patent
atpD


951
Streptococcus oralis
This patent
atpD


952
Streptococcus pneumoniae
This patent
atpD


953
Streptococcus pneumoniae
This patent
atpD


954
Streptococcus pneumoniae
This patent
atpD


955
Streptococcus pneumoniae
This patent
atpD


956
Babesia microti
This patent
atpD (V)


957
Entamoeba histolytica
This patent
atpD (V)


958
Fusobacterium nucleatum subsp. polymorphum
This patent
atpD (V)


959
Leishmania aethiopica
This patent
atpD (V)


960
Leishmania tropica
This patent
atpD (V)


961
Leishmania guyanensis
This patent
atpD (V)


962
Leishmania donovani
This patent
atpD (V)


963
Leishmania hertigi
This patent
atpD (V)


964
Leishmania mexicana
This patent
atpD (V)


965
Leishmania tropica
This patent
atpD (V)


966
Peptostreptococcus anaerobius
This patent
atpD (V)


967
Bordetella pertussis
This patent
tuf


968
Bordetella pertussis
This patent
tuf


969
Enterococcus columbae
This patent
tuf


970
Enterococcus flavescens
This patent
tuf


971
Streptococcus pneumoniae
This patent
tuf


972
Escherichia coli
This patent
tuf


973
Escherichia coli
This patent
tuf


974
Escherichia coli
This patent
tuf


975
Escherichia coli
This patent
tuf


976
Mycobacterium avium
This patent
tuf


977
Streptococcus pneumoniae
This patent
tuf


978
Mycobacterium gordonae
This patent
tuf


979
Streptococcus pneumoniae
This patent
tuf


980
Mycobacterium tuberculosis
This patent
tuf


981
Staphylococcus warneri
This patent
tuf


982
Streptococcus mitis
This patent
tuf


983
Streptococcus mitis
This patent
tuf


984
Streptococcus mitis
This patent
tuf


985
Streptococcus oralis
This patent
tuf


986
Streptococcus pneumoniae
This patent
tuf


987
Enterococcus hirae
This patent
tuf (C)


988
Enterococcus mundtii
This patent
tuf (C)


989
Enterococcus raffinosus
This patent
tuf (C)


990
Bacillus anthracis
This patent
recA


991
Prevotella melaninogenica
This patent
recA


992
Enterococcus casseliflavus
This patent
tuf


993
Streptococcus pyogenes
Database
speA


1002
Streptococcus pyogenes
WO98/20157
tuf


1003
Bacillus cereus
This patent
recA


1004
Streptococcus pneumoniae
This patent
pbp1a


1005
Streptococcus pneumoniae
This patent
pbp1a


1006
Streptococcus pneumoniae
This patent
pbp1a


1007
Streptococcus pneumoniae
This patent
pbp1a


1008
Streptococcus pneumoniae
This patent
pbp1a


1009
Streptococcus pneumoniae
This patent
pbp1a


1010
Streptococcus pneumoniae
This patent
pbp1a


1011
Streptococcus pneumoniae
This patent
pbp1a


1012
Streptococcus pneumoniae
This patent
pbp1a


1013
Streptococcus pneumoniae
This patent
pbp1a


1014
Streptococcus pneumoniae
This patent
pbp1a


1015
Streptococcus pneumoniae
This patent
pbp1a


1016
Streptococcus pneumoniae
This patent
pbp1a


1017
Streptococcus pneumoniae
This patent
pbp1a


1018
Streptococcus pneumoniae
This patent
pbp1a


1019
Streptococcus pneumoniae
This patent
pbp2b


1020
Streptococcus pneumoniae
This patent
pbp2b


1021
Streptococcus pneumoniae
This patent
pbp2b


1022
Streptococcus pneumoniae
This patent
pbp2b


1023
Streptococcus pneumoniae
This patent
pbp2b


1024
Streptococcus pneumoniae
This patent
pbp2b


1025
Streptococcus pneumoniae
This patent
pbp2b


1026
Streptococcus pneumoniae
This patent
pbp2b


1027
Streptococcus pneumoniae
This patent
pbp2b


1028
Streptococcus pneumoniae
This patent
pbp2b


1029
Streptococcus pneumoniae
This patent
pbp2b


1030
Streptococcus pneumoniae
This patent
pbp2b


1031
Streptococcus pneumoniae
This patent
pbp2b


1032
Streptococcus pneumoniae
This patent
pbp2b


1033
Streptococcus pneumoniae
This patent
pbp2b


1034
Streptococcus pneumoniae
This patent
pbp2x


1035
Streptococcus pneumoniae
This patent
pbp2x


1036
Streptococcus pneumoniae
This patent
pbp2x


1037
Streptococcus pneumoniae
This patent
pbp2x


1038
Streptococcus pneumoniae
This patent
pbp2x


1039
Streptococcus pneumoniae
This patent
pbp2x


1040
Streptococcus pneumoniae
This patent
pbp2x


1041
Streptococcus pneumoniae
This patent
pbp2x


1042
Streptococcus pneumoniae
This patent
pbp2x


1043
Streptococcus pneumoniae
This patent
pbp2x


1044
Streptococcus pneumoniae
This patent
pbp2x


1045
Streptococcus pneumoniae
This patent
pbp2x


1046
Streptococcus pneumoniae
This patent
pbp2x


1047
Streptococcus pneumoniae
This patent
pbp2x


1048
Streptococcus pneumoniae
This patent
pbp2x


1049
Enterococcus faecium
This patent
vanA


1050
Enterococcus gallinarum
This patent
vanA


1051
Enterococcus faecium
This patent
vanA


1052
Enterococcus faecium
This patent
vanA


1053
Enterococcus faecium
This patent
vanA


1054
Enterococcus faecalis
This patent
vanA


1055
Enterococcus gallinarum
This patent
vanA


1056
Enterococcus faecium
This patent
vanA


1057
Enterococcus flavescens
This patent
vanA


1058
Enterococcus gallinarum
This patent
vanC1


1059
Enterococcus gallinarum
This patent
vanC1


1060
Enterococcus casseliflavus
This patent
vanC2


1061
Enterococcus casseliflavus
This patent
vanC2


1062
Enterococcus casseliflavus
This patent
vanC2


1063
Enterococcus casseliflavus
This patent
vanC2


1064
Enterococcus flavescens
This patent
vanC3


1065
Enterococcus flavescens
This patent
vanC3


1066
Enterococcus flavescens
This patent
vanC3


1067
Enterococcus faecium
This patent
vanXY


1068
Enterococcus faecium
This patent
vanXY


1069
Enterococcus faecium
This patent
vanXY


1070
Enterococcus faecalis
This patent
vanXY


1071
Enterococcus gallinarum
This patent
vanXY


1072
Enterococcus faecium
This patent
vanXY


1073
Enterococcus flavescens
This patent
vanXY


1074
Enterococcus faecium
This patent
vanXY


1075
Enterococcus gallinarum
This patent
vanXY


1076
Escherichia coli
Database
stx1


1077
Escherichia coli
Database
stx2


1093
Staphylococcus saprophyticus
This patent
unknown


1117
Enterococcus faecium
Database
vanB


1138
Enterococcus gallinarum
Database
vanC1


1139
Enterococcus faecium
Database
vanA


1140
Enterococcus casseliflavus
Database
vanC2


1141
Enterococcus faecium
Database
vanHAXY


1169
Streptococcus pneumoniae
Database
pbp1a


1172
Streptococcus pneumoniae
Database
pbp2b


1173
Streptococcus pneumoniae
Database
pbp2x


1178
Staphylococcus aureus
Database
mecA


1183
Streptococcus pneumoniae
Database
hexA


1184
Streptococcus pneumoniae
This patent
hexA


1185
Streptococcus pneumoniae
This patent
hexA


1186
Streptococcus pneumoniae
This patent
hexA


1187
Streptococcus pneumoniae
This patent
hexA


1188
Streptococcus oralis
This patent
hexA


1189
Streptococcus mitis
This patent
hexA


1190
Streptococcus mitis
This patent
hexA


1191
Streptococcus mitis
This patent
hexA


1198
Staphylococcus saprophyticus
This patent
unknown


1215
Streptococcus pyogenes
Database
pcp


1230
Escherichia coli
Database
tuf (EF-G)


1242
Enterococcus faecium
Database
ddl


1243
Enterococcus faecalis
Database
mtlF, mtlD


1244
Staphylococcus aureus subsp. aureus
This patent
unknown


1245
Bacillus anthracis
This patent
atpD


1246
Bacillus mycoides
This patent
atpD


1247
Bacillus thuringiensis
This patent
atpD


1248
Bacillus thuringiensis
This patent
atpD


1249
Bacillus thuringiensis
This patent
atpD


1250
Bacillus weihenstephanensis
This patent
atpD


1251
Bacillus thuringiensis
This patent
atpD


1252
Bacillus thuringiensis
This patent
atpD


1253
Bacillus cereus
This patent
atpD


1254
Bacillus cereus
This patent
atpD


1255
Staphylococcus aureus
This patent
gyrA


1256
Bacillus weihenstephanensis
This patent
atpD


1257
Bacillus anthracis
This patent
atpD


1258
Bacillus thuringiensis
This patent
atpD


1259
Bacillus cereus
This patent
atpD


1260
Bacillus cereus
This patent
atpD


1261
Bacillus thuringiensis
This patent
atpD


1262
Bacillus thuringiensis
This patent
atpD


1263
Bacillus thuringiensis
This patent
atpD


1264
Bacillus thuringiensis
This patent
atpD


1265
Bacillus anthracis
This patent
atpD


1266
Paracoccidioides brasiliensis
This patent
tuf (EF-1)


1267
Blastomyces dermatitidis
This patent
tuf (EF-1)


1268
Histoplasma capsulatum
This patent
tuf (EF-1)


1269
Trichophyton rubrum
This patent
tuf (EF-1)


1270
Microsporum canis
This patent
tuf (EF-1)


1271
Aspergillus versicolor
This patent
tuf (EF-1)


1272
Exophiala moniliae
This patent
tuf (EF-1)


1273
Hortaea wemeckii
This patent
tuf (EF-1)


1274
Fusarium solani
This patent
tuf (EF-1)


1275
Aureobasidium pullulans
This patent
tuf (EF-1)


1276
Blastomyces dermatitidis
This patent
tuf (EF-1)


1277
Exophiala dermatitidis
This patent
tuf (EF-1)


1278
Fusarium moniliforme
This patent
tuf (EF-1)


1279
Aspergillus terreus
This patent
tuf (EF-1)


1280
Aspergillus fumigatus
This patent
tuf (EF-1)


1281
Cryptococcus laurentii
This patent
tuf (EF-1)


1282
Emmonsia parva
This patent
tuf (EF-1)


1283
Fusarium solani
This patent
tuf (EF-1)


1284
Sporothrix schenckii
This patent
tuf (EF-1)


1285
Aspergillus nidulans
This patent
tuf (EF-1)


1286
Cladophialophora carrionii
This patent
tuf (EF-1)


1287
Exserohilum rostratum
This patent
tuf (EF-1)


1288
Bacillus thuringiensis
This patent
recA


1289
Bacillus thuringiensis
This patent
recA


1299
Staphylococcus aureus
Database
gyrA


1300
Escherichia coli
Database
gyrA


1307
Staphylococcus aureus
Database
gyrB


1320
Escherichia coli
Database
parC (grlA)


1321
Staphylococcus aureus
Database
parC (grlA)


1328
Staphylococcus aureus
Database
parE (grlB)


1348
unidentified bacterium
Database
aac2Ia


1351
Pseudomonas aeruginosa
Database
aac3Ib


1356
Serratia marcescens
Database
aac3IIb


1361
Escherichia coli
Database
aac3IVa


1366
Enterobacter cloacae
Database
aac3VIa


1371
Citrobacter koseri
Database
aac6Ia


1376
Serratia marcescens
Database
aac6Ic


1381
Escherichia coli
Database
ant3Ia


1386
Staphylococcus aureus
Database
ant4Ia


1391
Escherichia coli
Database
aph3Ia


1396
Escherichia coli
Database
aph3IIa


1401
Enterococcus faecalis
Database
aph3IIIa


1406
Acinetobacter baumannii
Database
aph3VIa


1411
Pseudomonas aeruginosa
Database
blaCARB


1416
Klebsiella pneumoniae
Database
blaCMY-2


1423
Escherichia coli
Database
blaCTX-M-1


1428
Salmonella choleraesuis subsp. choleraesuis serotype
Database
blaCTX-M-2



Typhimurium


1433
Pseudomonas aeruginosa
Database
blaIMP


1438
Escherichia coli
Database
blaOXA2


1439
Pseudomonas aeruginosa
Database
blaOXA10


1442
Pseudomonas aeruginosa
Database
blaPER1


1445
Salmonella choleraesuis subsp. choleraesuis serotype
Database
blaPER2



Typhimurium


1452
Staphylococcus epidermidis
Database
dfrA


1461
Escherichia coli
Database
dhfrIa


1470
Escherichia coli
Database
dhfrIb


1475
Escherichia coli
Database
dhfrV


1480
Proteus mirabilis
Database
dhfrVI


1489
Escherichia coli
Database
dhfrVII


1494
Escherichia coli
Database
dhfrVIII


1499
Escherichia coli
Database
dhfrIX


1504
Escherichia coli
Database
dhfrXII


1507
Escherichia coli
Database
dhfrXIII


1512
Escherichia coli
Database
dhfrXV


1517
Escherichia coli
Database
dhfrXVII


1518
Acinetobacter lwoffii
This patent
fusA


1519
Acinetobacter lwoffii
This patent
fusA-tuf spacer


1520
Acinetobacter lwoffii
This patent
tuf


1521
Haemophilus influenzae
This patent
fusA


1522
Haemophilus influenzae
This patent
fusA-tuf spacer


1523
Haemophilus influenzae
This patent
tuf


1524
Proteus mirabilis
This patent
fusA


1525
Proteus mirabilis
This patent
fusA-tuf spacer


1526
Proteus mirabilis
This patent
tuf


1527
Campylobacter curvus
This patent
atpD


1530
Escherichia coli
Database
ereA


1535
Escherichia coli
Database
ereB


1540
Staphylococcus haemolyticus
Database
linA


1545
Enterococcus faecium
Database
linB


1548
Streptococcus pyogenes
Database
mefA


1551
Streptococcus pneumoniae
Database
mefE


1560
Escherichia coli
Database
mphA


1561
Candida albicans
This patent
tuf (EF-1)


1562
Candida dubliniensis
This patent
tuf (EF-1)


1563
Candida famata
This patent
tuf (EF-1)


1564
Candida glabrata
This patent
tuf (EF-1)


1565
Candida guilliermondii
This patent
tuf (EF-1)


1566
Candida haemulonii
This patent
tuf (EF-1)


1567
Candida kefyr
This patent
tuf (EF-1)


1568
Candida lusitaniae
This patent
tuf (EF-1)


1569
Candida sphaerica
This patent
tuf (EF-1)


1570
Candida tropicalis
This patent
tuf (EF-1)


1571
Candida viswanathii
This patent
tuf (EF-1)


1572
Alcaligenes faecalis subsp. faecalis
This patent
tuf


1573
Prevotella buccalis
This patent
tuf


1574
Succinivibrio dextrinosolvens
This patent
tuf


1575
Tetragenococcus halophilus
This patent
tuf


1576
Campylobacter jejuni subsp. jejuni
This patent
atpD


1577
Campylobacter rectus
This patent
atpD


1578
Enterococcus casseliflavus
This patent
fusA


1579
Enterococcus gallinarum
This patent
fusA


1580
Streptococcus mitis
This patent
fusA


1585
Enterococcus faecium
Database
satG


1590
Cloning vector pFW16
Database
tetM


1594
Enterococcus faecium
Database
vanD


1599
Enterococcus faecalis
Database
vanE


1600
Campylobacter jejuni subsp. doylei
This patent
atpD


1601
Enterococcus sulfureus
This patent
atpD


1602
Enterococcus solitarius
This patent
atpD


1603
Campylobacter sputorum subsp. sputorum
This patent
atpD


1604
Enterococcus pseudoavium
This patent
atpD


1607
Klebsiella omithinolytica
This patent
gyrA


1608
Klebsiella oxytoca
This patent
gyrA


1613
Staphylococcus aureus
Database
vatB


1618
Staphylococcus cohnii
Database
vatC


1623
Staphylococcus aureus
Database
vga


1628
Staphylococcus aureus
Database
vgaB


1633
Staphylococcus aureus
Database
vgb


1638
Aspergillus fumigatus
This patent
atpD


1639
Aspergillus fumigatus
This patent
atpD


1640
Bacillus mycoides
This patent
atpD


1641
Bacillus mycoides
This patent
atpD


1642
Bacillus mycoides
This patent
atpD


1643
Bacillus pseudomycoides
This patent
atpD


1644
Bacillus pseudomycoides
This patent
atpD


1645
Budvicia aquatica
This patent
atpD


1646
Buttiauxella agrestis
This patent
atpD


1647
Candida norvegica
This patent
atpD


1648
Streptococcus pneumoniae
This patent
pbp1a


1649
Campylobacter lari
This patent
atpD


1650
Coccidioides immitis
This patent
atpD


1651
Emmonsia parva
This patent
atpD


1652
Erwinia amylovora
This patent
atpD


1653
Fonsecaea pedrosoi
This patent
atpD


1654
Fusarium moniliforme
This patent
atpD


1655
Klebsiella oxytoca
This patent
atpD


1656
Microsporum audouinii
This patent
atpD


1657
Obesumbacterium proteus
This patent
atpD


1658
Paracoccidioides brasiliensis
This patent
atpD


1659
Plesiomonas shigelloides
This patent
atpD


1660
Shewanella putrefaciens
This patent
atpD


1662
Campylobacter curvus
This patent
tuf


1663
Campylobacter rectus
This patent
tuf


1664
Fonsecaea pedrosoi
This patent
tuf


1666
Microsporum audouinii
This patent
tuf


1667
Piedraia hortai
This patent
tuf


1668
Escherichia coli
Database
tuf


1669
Saksenaea vasiformis
This patent
tuf


1670
Trichophyton tonsurans
This patent
tuf


1671
Enterobacter aerogenes
This patent
atpD


1672
Bordetella pertussis
Database
atpD


1673
Arcanobacterium haemolyticum
This patent
tuf


1674
Butyrivibrio fibrisolvens
This patent
tuf


1675
Campylobacter jejuni subsp. doylei
This patent
tuf


1676
Campylobacter lari
This patent
tuf


1677
Campylobacter sputorum subsp. sputorum
This patent
tuf


1678
Campylobacter upsaliensis
This patent
tuf


1679
Globicatella sanguis
This patent
tuf


1680
Lactobacillus acidophilus
This patent
tuf


1681
Leuconostoc mesenteroides subsp. dextranicum
This patent
tuf


1682
Prevotella buccalis
This patent
tuf


1683
Ruminococcus bromii
This patent
tuf


1684
Paracoccidioides brasiliensis
This patent
atpD


1685
Candida norvegica
This patent
tuf (EF-1)


1686
Aspergillus nidulans
This patent
tuf


1687
Aspergillus terreus
This patent
tuf


1688
Candida norvegica
This patent
tuf


1689
Candida parapsilosis
This patent
tuf


1702
Streptococcus gordonii
WO98/20157
recA


1703
Streptococcus mutans
WO98/20157
recA


1704
Streptococcus pneumoniae
WO98/20157
recA


1705
Streptococcus pyogenes
WO98/20157
recA


1706
Streptococcus salivarius subsp. thermophilus
WO98/20157
recA


1707
Escherichia coli
WO98/20157
oxa


1708
Enterococcus faecalis
WO98/20157
blaZ


1709
Pseudomonas aeruginosa
WO98/20157
aac6′-IIa


1710
Staphylococcus aureus
WO98/20157
ermA


1711
Escherichia coli
WO98/20157
ermB


1712
Staphylococcus aureus
WO98/20157
ermC


1713
Enterococcus faecalis
WO98/20157
vanB


1714
Campylobacter jejuni subsp. jejuni
This patent
recA


1715
Abiotrophia adiacens
WO98/20157
tuf


1716
Abiotrophia defectiva
WO98/20157
tuf


1717
Corynebacterium accolens
WO98/20157
tuf


1718
Corynebacterium genitalium
WO98/20157
tuf


1719
Corynebacterium jeikeium
WO98/20157
tuf


1720
Corynebacterium pseudodiphtheriticum
WO98/20157
tuf


1721
Corynebacterium striatum
WO98/20157
tuf


1722
Enterococcus avium
WO98/20157
tuf


1723
Gardnerella vaginalis
WO98/20157
tuf


1724
Listeria innocua
WO98/20157
tuf


1725
Listeria ivanovii
WO98/20157
tuf


1726
Listeria monocytogenes
WO98/20157
tuf


1727
Listeria seeligeri
WO98/20157
tuf


1728
Staphylococcus aureus
WO98/20157
tuf


1729
Staphylococcus saprophyticus
WO98/20157
tuf


1730
Staphylococcus simulans
WO98/20157
tuf


1731
Streptococcus agalactiae
WO98/20157
tuf


1732
Streptococcus pneumoniae
WO98/20157
tuf


1733
Streptococcus salivarius
WO98/20157
tuf


1734
Agrobacterium radiobacter
WO98/20157
tuf


1735
Bacillus subtilis
WO98/20157
tuf


1736
Bacteroides fragilis
WO98/20157
tuf


1737
Borrelia burgdorferi
WO98/20157
tuf


1738
Brevibacterium linens
WO98/20157
tuf


1739
Chlamydia trachomatis
WO98/20157
tuf


1740
Fibrobacter succinogenes
WO98/20157
tuf


1741
Flavobacterium ferrugineum
WO98/20157
tuf


1742
Helicobacter pylori
WO98/20157
tuf


1743
Micrococcus luteus
WO98/20157
tuf


1744
Mycobacterium tuberculosis
WO98/20157
tuf


1745
Mycoplasma genitalium
WO98/20157
tuf


1746
Neisseria gonorrhoeae
WO98/20157
tuf


1747
Rickettsia prowazekii
WO98/20157
tuf


1748
Salmonella choleraesuis subsp. choleraesuis
WO98/20157
tuf



serotype Typhimurium


1749
Shewanella putrefaciens
WO98/20157
tuf


1750
Stigmatella aurantiaca
WO98/20157
tuf


1751
Thiomonas cuprina
WO98/20157
tuf


1752
Treponema pallidum
WO98/20157
tuf


1753
Ureaplasma urealyticum
WO98/20157
tuf


1754
Wolinella succinogenes
WO98/20157
tuf


1755
Burkholderia cepacia
WO98/20157
tuf


1756
Bacillus anthracis
This patent
recA


1757
Bacillus anthracis
This patent
recA


1758
Bacillus cereus
This patent
recA


1759
Bacillus cereus
This patent
recA


1760
Bacillus mycoides
This patent
recA


1761
Bacillus pseudomycoides
This patent
recA


1762
Bacillus thuringiensis
This patent
recA


1763
Bacillus thuringiensis
This patent
recA


1764
Klebsiella oxytoca
This patent
gyrA


1765
Klebsiella pneumoniae subsp. ozaenae
This patent
gyrA


1766
Klebsiella planticola
This patent
gyrA


1767
Klebsiella pneumoniae
This patent
gyrA


1768
Klebsiella pneumoniae subsp. pneumoniae
This patent
gyrA


1769
Klebsiella pneumoniae subsp. pneumoniae
This patent
gyrA


1770
Klebsiella pneumoniae subsp. rhinoscleromatis
This patent
gyrA


1771
Klebsiella terrigena
This patent
gyrA


1772
Legionella pneumophila subsp. pneumophila
This patent
gyrA


1773
Proteus mirabilis
This patent
gyrA


1774
Providencia rettgeri
This patent
gyrA


1775
Proteus vulgaris
This patent
gyrA


1776
Yersinia enterocolitica
This patent
gyrA


1777
Klebsiella oxytoca
This patent
parC (grlA)


1778
Klebsiella oxytoca
This patent
parC (grlA)


1779
Klebsiella pneumoniae subsp. ozaenae
This patent
parC (grlA)


1780
Klebsiella planticola
This patent
parC (grlA)


1781
Klebsiella pneumoniae
This patent
parC (grlA)


1782
Klebsiella pneumoniae subsp. pneumoniae
This patent
parC (grlA)


1783
Klebsiella pneumoniae subsp. pneumoniae
This patent
parC (grlA)


1784
Klebsiella pneumoniae subsp. rhinoscleromatis
This patent
parC (grlA)


1785
Klebsiella terrigena
This patent
parC (grlA)


1786
Bacillus cereus
This patent
fusA


1787
Bacillus cereus
This patent
fusA


1788
Bacillus anthracis
This patent
fusA


1789
Bacillus cereus
This patent
fusA


1790
Bacillus anthracis
This patent
fusA


1791
Bacillus pseudomycoides
This patent
fusA


1792
Bacillus cereus
This patent
fusA


1793
Bacillus anthracis
This patent
fusA


1794
Bacillus cereus
This patent
fusA


1795
Bacillus weihenstephanensis
This patent
fusA


1796
Bacillus mycoides
This patent
fusA


1797
Bacillus thuringiensis
This patent
fusA


1798
Bacillus weihenstephanensis
This patent
fusA-tuf spacer


1799
Bacillus thuringiensis
This patent
fusA-tuf spacer


1800
Bacillus anthracis
This patent
fusA-tuf spacer


1801
Bacillus pseudomycoides
This patent
fusA-tuf spacer


1802
Bacillus anthracis
This patent
fusA-tuf spacer


1803
Bacillus cereus
This patent
fusA-tuf spacer


1804
Bacillus cereus
This patent
fusA-tuf spacer


1805
Bacillus mycoides
This patent
fusA-tuf spacer


1806
Bacillus cereus
This patent
fusA-tuf spacer


1807
Bacillus cereus
This patent
fusA-tuf spacer


1808
Bacillus cereus
This patent
fusA-tuf spacer


1809
Bacillus anthracis
This patent
fusA-tuf spacer


1810
Bacillus mycoides
This patent
tuf


1811
Bacillus thuringiensis
This patent
tuf


1812
Bacillus cereus
This patent
tuf


1813
Bacillus weihenstephanensis
This patent
tuf


1814
Bacillus anthracis
This patent
tuf


1815
Bacillus cereus
This patent
tuf


1816
Bacillus cereus
This patent
tuf


1817
Bacillus anthracis
This patent
tuf


1818
Bacillus cereus
This patent
tuf


1819
Bacillus anthracis
This patent
tuf


1820
Bacillus pseudomycoides
This patent
tuf


1821
Bacillus cereus
This patent
tuf


1822
Streptococcus oralis
This patent
fusA


1823
Budvicia aquatica
This patent
fusA


1824
Buttiauxella agrestis
This patent
fusA


1825
Klebsiella oxytoca
This patent
fusA


1826
Plesiomonas shigelloides
This patent
fusA


1827
Shewanella putrefaciens
This patent
fusA


1828
Obesumbacterium proteus
This patent
fusA


1829
Klebsiella oxytoca
This patent
fusA-tuf spacer


1830
Budvicia aquatica
This patent
fusA-tuf spacer


1831
Plesiomonas shigelloides
This patent
fusA-tuf spacer


1832
Obesumbacterium proteus
This patent
fusA-tuf spacer


1833
Shewanella putrefaciens
This patent
fusA-tuf spacer


1834
Buttiauxella agrestis
This patent
fusA-tuf spacer


1835
Campylobacter coli
This patent
tuf


1836
Campylobacter fetus subsp. fetus
This patent
tuf


1837
Campylobacter fetus subsp. venerealis
This patent
tuf


1838
Buttiauxella agrestis
This patent
tuf


1839
Klebsiella oxytoca
This patent
tuf


1840
Plesiomonas shigelloides
This patent
tuf


1841
Shewanella putrefaciens
This patent
tuf


1842
Obesumbacterium proteus
This patent
tuf


1843
Budvicia aquatica
This patent
tuf


1844
Abiotrophia adiacens
This patent
atpD


1845
Arcanobacterium haemolyticum
This patent
atpD


1846
Basidiobolus ranarum
This patent
atpD


1847
Blastomyces dermatitidis
This patent
atpD


1848
Blastomyces dermatitidis
This patent
atpD


1849
Campylobacter coli
This patent
atpD


1850
Campylobacter fetus subsp. fetus
This patent
atpD


1851
Campylobacter fetus subsp. venerealis
This patent
atpD


1852
Campylobacter gracilis
This patent
atpD


1853
Campylobacter jejuni subsp. jejuni
This patent
atpD


1854
Enterococcus cecorum
This patent
atpD


1855
Enterococcus columbae
This patent
atpD


1856
Enterococcus dispar
This patent
atpD


1857
Enterococcus malodoratus
This patent
atpD


1858
Enterococcus mundtii
This patent
atpD


1859
Enterococcus raffinosus
This patent
atpD


1860
Globicatella sanguis
This patent
atpD


1861
Lactococcus garvieae
This patent
atpD


1862
Lactococcus lactis
This patent
atpD


1863
Listeria ivanovii
This patent
atpD


1864
Succinivibrio dextrinosolvens
This patent
atpD


1865
Tetragenococcus halophilus
This patent
atpD


1866
Campylobacter fetus subsp. fetus
This patent
recA


1867
Campylobacter fetus subsp. venerealis
This patent
recA


1868
Campylobacter jejuni subsp. jejuni
This patent
recA


1869
Enterococcus avium
This patent
recA


1870
Enterococcus faecium
This patent
recA


1871
Listeria monocytogenes
This patent
recA


1872
Streptococcus mitis
This patent
recA


1873
Streptococcus oralis
This patent
recA


1874
Aspergillus fumigatus
This patent
tuf (M)


1875
Aspergillus versicolor
This patent
tuf (M)


1876
Basidiobolus ranarum
This patent
tuf (M)


1877
Campylobacter gracilis
This patent
tuf


1878
Campylobacter jejuni subsp. jejuni
This patent
tuf


1879
Coccidioides immitis
This patent
tuf (M)


1880
Erwinia amylovora
This patent
tuf


1881
Salmonella choleraesuis subsp. choleraesuis serotype
This patent
tuf



Typhimurium


1899
Klebsiella pneumoniae
Database
blaSHV


1900
Klebsiella pneumoniae
Database
blaSHV


1901
Escherichia coli
Database
blaSHV


1902
Klebsiella pneumoniae
Database
blaSHV


1903
Klebsiella pneumoniae
Database
blaSHV


1904
Escherichia coli
Database
blaSHV


1905
Pseudomonas aeruginosa
Database
blaSHV


1927
Neisseria meningitidis
Database
blaTEM


1928
Escherichia coli
Database
blaTEM


1929
Klebsiella oxytoca
Database
blaTEM


1930
Escherichia coli
Database
blaTEM


1931
Escherichia coli
Database
blaTEM


1932
Escherichia coli
Database
blaTEM


1933
Escherichia coli
Database
blaTEM


1954
Klebsiella pneumoniae subsp. pneumoniae
Database
gyrA


1956
Candida inconspicua
This patent
tuf (M)


1957
Candida utilis
This patent
tuf (M)


1958
Candida zeylanoides
This patent
tuf (M)


1959
Candida catenulata
This patent
tuf (M)


1960
Candida krusei
This patent
tuf (M)


1965
Plasmid pGS05
Database
sulII


1970
Transposon Tn10
Database
tetB


1985
Cryptococcus neoformans
Database
tuf (EF-1)


1986
Cryptococcus neoformans
Database
tuf (EF-1)


1987
Saccharomyces cerevisiae
Database
tuf (EF-1)


1988
Saccharomyces cerevisiae
Database
tuf (EF-1)


1989
Eremothecium gossypii
Database
tuf (EF-1)


1990
Eremothecium gossypii
Database
tuf (EF-1)


1991
Aspergillus oryzae
Database
tuf (EF-1)


1992
Aureobasidium pullulans
Database
tuf (EF-1)


1993
Histoplasma capsulatum
Database
tuf (EF-1)


1994
Neurospora crassa
Database
tuf (EF-1)


1995
Podospora anserina
Database
tuf (EF-1)


1996
Podospora curvicolla
Database
tuf (EF-1)


1997
Sordaria macrospora
Database
tuf (EF-1)


1998
Trichoderma reesei
Database
tuf (EF-1)


2004
Candida albicans
Database
tuf (M)


2005
Schizosaccharomyces pombe
Database
tuf (M)


2010
Klebsiella pneumoniae
Database
blaTEM


2011
Klebsiella pneumoniae
Database
blaTEM


2013
Kluyvera ascorbata
This patent
gyrA


2014
Klvyvera georgiana
This patent
gyrA


2047
Streptococcus pneumoniae
Database
pbp1A


2048
Streptococcus pneumoniae
Database
pbp1A


2049
Streptococcus pneumoniae
Database
pbp1A


2050
Streptococcus pneumoniae
Database
pbp1A


2051
Streptococcus pneumoniae
Database
pbp1A


2052
Streptococcus pneumoniae
Database
pbp1A


2053
Streptococcus pneumoniae
Database
pbp1A


2054
Streptococcus pneumoniae
Database
gyrA


2055
Streptococcus pneumoniae
Database
parC


2056
Streptococcus pneumoniae
This patent
pbp1A


2057
Streptococcus pneumoniae
This patent
pbp1A


2058
Streptococcus pneumoniae
This patent
pbp1A


2059
Streptococcus pneumoniae
This patent
pbp1A


2060
Streptococcus pneumoniae
This patent
pbp1A


2061
Streptococcus pneumoniae
This patent
pbp1A


2062
Streptococcus pneumoniae
This patent
pbp1A


2063
Streptococcus pneumoniae
This patent
pbp1A


2064
Streptococcus pneumoniae
This patent
pbp1A


2072
Mycobacterium tuberculosis
Database
rpoB


2097
Mycoplasma pneumoniae
Database
tuf


2101
Mycobacterium tuberculosis
Database
inhA


2105
Mycobacterium tuberculosis
Database
embB


2129
Clostridium difficile
Database
cdtA


2130
Clostridium difficile
Database
cdtB


2137
Pseudomonas putida
Genome project
tuf


2138
Pseudomonas aeruginosa
Genome project
tuf


2139
Campylobacter jejuni
Database
atpD


2140
Streptococcus pneumoniae
Database
pbp1a


2144
Staphylococcus aureus
Database
mupA


2147
Escherichia coli
Database
catI


2150
Escherichia coli
Database
catII


2153
Shigella flexneri
Database
catIII


2156
Clostridium perfringens
Database
catP


2159
Staphylococcus aureus
Database
cat


2162
Staphylococcus aureus
Database
cat


2165
Salmonella typhimurium
Database
ppflo-like


2183
Alcaligenes faecalis subsp. faecalis
This patent
tuf


2184
Campylobacter coli
This patent
fusA


2185
Succinivibrio dextrinosolvens
This patent
tuf


2186
Tetragenococcus halophilus
This patent
tuf


2187
Campylobacter jejuni subsp. jejuni
This patent
fusA


2188
Campylobacter jejuni subsp. jejuni
This patent
fusA


2189
Leishmania guyanensis
This patent
atpD


2190
Trypanosoma brucei brucei
This patent
atpD


2191
Aspergillus nidulans
This patent
atpD


2192
Leishmania panamensis
This patent
atpD


2193
Aspergillus nidulans
This patent
tuf (M)


2194
Aureobasidium pullulans
This patent
tuf (M)


2195
Emmonsia parva
This patent
tuf (M)


2196
Exserohilum rostratum
This patent
tuf (M)


2197
Fusarium moniliforme
This patent
tuf (M)


2198
Fusarium solani
This patent
tuf (M)


2199
Histoplasma capsulatum
This patent
tuf (M)


2200
Kocuria kristinae
This patent
tuf


2201
Vibrio mimicus
This patent
tuf


2202
Citrobacter freundii
This patent
recA


2203
Clostridium botulinum
This patent
recA


2204
Francisella tularensis
This patent
recA


2205
Peptostreptococcus anaerobius
This patent
recA


2206
Peptostreptococcus asaccharolyticus
This patent
recA


2207
Providencia stuartii
This patent
recA


2208
Salmonella choleraesuis subsp. choleraesuis
This patent
recA



serotype Paratyphi A


2209
Salmonella choleraesuis subsp. choleraesuis
This patent
recA



serotype Typhimurium


2210
Staphylococcus saprophyticus
This patent
recA


2211
Yersinia pseudotuberculosis
This patent
recA


2212
Zoogloea ramigera
This patent
recA


2214
Abiotrophia adiacens
This patent
fusA


2215
Acinetobacter baumannii
This patent
fusA


2216
Actinomyces meyeri
This patent
fusA


2217
Clostridium difficile
This patent
fusA


2218
Corynebacterium diphtheriae
This patent
fusA


2219
Enterobacter cloacae
This patent
fusA


2220
Klebsiella pneumoniae subsp. pneumoniae
This patent
fusA


2221
Listeria monocytogenes
This patent
fusA


2222
Mycobacterium avium
This patent
fusA


2223
Mycobacterium gordonae
This patent
fusA


2224
Mycobacterium kansasii
This patent
fusA


2225
Mycobacterium terrae
This patent
fusA


2226
Neisseria polysaccharea
This patent
fusA


2227
Staphylococcus epidermidis
This patent
fusA


2228
Staphylococcus haemolyticus
This patent
fusA


2229
Succinivibrio dextrinosolvens
This patent
fusA


2230
Tetragenococcus halophilus
This patent
fusA


2231
Veillonella parvula
This patent
fusA


2232
Yersinia pseudotuberculosis
This patent
fusA


2233
Zoogloea ramigera
This patent
fusA


2234
Aeromonas hydrophila
This patent
fusA


2235
Abiotrophia adiacens
This patent
fusA-tuf spacer


2236
Acinetobacter baumannii
This patent
fusA-tuf spacer


2237
Actinomyces meyeri
This patent
fusA-tuf spacer


2238
Clostridium difficile
This patent
fusA-tuf spacer


2239
Corynebacterium diphtheriae
This patent
fusA-tuf spacer


2240
Enterobacter cloacae
This patent
fusA-tuf spacer


2241
Klebsiella pneumoniae subsp. pneumoniae
This patent
fusA-tuf spacer


2242
Listeria monocytogenes
This patent
fusA-tuf spacer


2243
Mycobacterium avium
This patent
fusA-tuf spacer


2244
Mycobacterium gordonae
This patent
fusA-tuf spacer


2245
Mycobacterium kansasii
This patent
fusA-tuf spacer


2246
Mycobacterium terrae
This patent
fusA-tuf spacer


2247
Neisseria polysaccharea
This patent
fusA-tuf spacer


2248
Staphylococcus epidermidis
This patent
fusA-tuf spacer


2249
Staphylococcus haemolyticus
This patent
fusA-tuf spacer


2255
Abiotrophia adiacens
This patent
tuf


2256
Acinetobacter baumannii
This patent
tuf


2257
Actinomyces meyeri
This patent
tuf


2258
Clostridium difficile
This patent
tuf


2259
Corynebacterium diphtheriae
This patent
tuf


2260
Enterobacter cloacae
This patent
tuf


2261
Klebsiella pneumoniae subsp. pneumoniae
This patent
tuf


2262
Listeria monocytogenes
This patent
tuf


2263
Mycobacterium avium
This patent
tuf


2264
Mycobacterium gordonae
This patent
tuf


2265
Mycobacterium kansasii
This patent
tuf


2266
Mycobacterium terrae
This patent
tuf


2267
Neisseria polysaccharea
This patent
tuf


2268
Staphylococcus epidermidis
This patent
tuf


2269
Staphylococcus haemolyticus
This patent
tuf


2270
Aeromonas hydrophila
This patent
tuf


2271
Bilophila wadsworthia
This patent
tuf


2272
Brevundimonas diminuta
This patent
tuf


2273
Streptococcus mitis
This patent
pbp1a


2274
Streptococcus mitis
This patent
pbp1a


2275
Streptococcus mitis
This patent
pbp1a


2276
Streptococcus oralis
This patent
pbp1a


2277
Escherichia coli
This patent
gyrA


2278
Escherichia coli
This patent
gyrA


2279
Escherichia coli
This patent
gyrA


2280
Escherichia coli
This patent
gyrA


2288
Enterococcus faecium
Database
ddI


2293
Enterococcus faecium
Database
vanA


2296
Enterococcus faecalis
Database
vanB





*tuf indicates tuf sequences, tuf (C) indicates tuf sequences divergent from main (usually A and B) copies of the elongation factor-Tu, tuf (EF-1) indicates tuf sequences of the eukaryotic type (elongation factor 1α), tuf (M) indicates tuf sequences from organellar (mostly mitochondrial) origin. fusA indicates fusA sequences; fusA-tuf spacer indicates the intergenic region between fusA and tuf. atpD indicates atpD sequences ot the F-type, atpD (V) indicates atpD sequences of the V-type. recA indicates recA sequences, recA(Rad51) indicates rad51 sequences or homologs and recA(Dmc1) indicates dmc1 sequences or homologs.













TABLE 8







Bacterial species used to test the specificity of the Streptococcus


agalactiae-specific amplification primers derived from tuf sequences.










Strain
Reference number







Streptococcus acidominimus
ATCC 51726



Streptococcus agalactiae
ATCC 12403



Streptococcus agalactiae
ATCC 12973



Streptococcus agalactiae
ATCC 13813



Streptococcus agalactiae
ATCC 27591



Streptococcus agalactiae
CDCs 1073



Streptococcus anginosus
ATCC 27335



Streptococcus anginosus
ATCC 33397



Streptococcus bovis
ATCC 33317



Streptococcus anginosus
ATCC 27823



Streptococcus cricetus
ATCC 19642



Streptococcus cristatus
ATCC 51100



Streptococcus downei
ATCC 33748



Streptococcus dysgalactiae
ATCC 43078



Streptococcus equi subsp. equi
ATCC 9528



Streptococcus ferus
ATCC 33477



Streptococcus gordonii
ATCC 10558



Streptococcus macacae
ATCC 35911



Streptococcus mitis
ATCC 49456



Streptococcus mutans
ATCC 25175



Streptococcus oralis
ATCC 35037



Streptococcus parasanguinis
ATCC 15912



Streptococcus parauberis
DSM 6631



Streptococcus pneumoniae
ATCC 27336



Streptococcus pyogenes
ATCC 19615



Streptococcus ratti
ATCC 19645



Streptococcus salivarius
ATCC 7073



Streptococcus sanguinis
ATCC 10556



Streptococcus sobrinus
ATCC 27352



Streptococcus suis
ATCC 43765



Streptococcus uberis
ATCC 19436



Streptococcus vestubularis
ATCC 49124



Bacteroides caccae
ATCC 43185



Bacteroides vulgatus
ATCC 8482



Bacteroides fragilis
ATCC 25285



Candida albicans
ATCC 11006



Clostridium innoculum
ATCC 14501



Clostridium ramosum
ATCC 25582



Lactobacillus casei subsp. casei
ATCC 393



Clostridium septicum
ATCC 12464



Corynebacterium cervicis
NCTC 10604



Corynebacterium genitalium
ATCC 33031



Corynebacterium urealyticum
ATCC 43042



Enterococcus faecalis
ATCC 29212



Enterococcus faecium
ATCC 19434



Eubacterium lentum
ATCC 43055



Eubacterium nodutum
ATCC 33099



Gardnerella vaginalis
ATCC 14018



Lactobacillus acidophilus
ATCC 4356



Lactobacillus crispatus
ATCC 33820



Lactobacillus gasseri
ATCC 33323



Lactobacillus johnsonii
ATCC 33200



Lactococcus lactis subsp. lactis
ATCC 19435



Lactococcus lactis subsp. lactis
ATCC 11454



Listeria innocua
ATCC 33090



Micrococcus luteus
ATCC 9341



Escherichia coli
ATCC 25922



Micrococcus lylae
ATCC 27566



Porphyromonas asaccharolytica
ATCC 25260



Prevotella corporis
ATCC 33547



Prevotella melanogenica
ATCC 25845



Staphylococcus aureus
ATCC 13301



Staphylococcus epidermidis
ATCC 14990



Staphylococcus saprophyticus
ATCC 15305

















TABLE 9







Bacterial species used to test the specificity of the Streptococcus


agalactiae-specific amplification primers derived from atpD sequences.










Strain
Reference number







Streptococcus acidominimus
ATCC 51726



Streptococcus agalactiae
ATCC 12400



Streptococcus agalactiae
ATCC 12403



Streptococcus agalactiae
ATCC 12973



Streptococcus agalactiae
ATCC 13813



Streptococcus agalactiae
ATCC 27591



Streptococcus agalactiae
CDCs-1073



Streptococcus anginosus
ATCC 27335



Streptococcus anginosus
ATCC 27823



Streptococcus bovis
ATCC 33317



Streptococcus cricetus
ATCC 19642



Streptococcus cristatus
ATCC 51100



Streptococcus downei
ATCC 33748



Streptococcus dysgalactiae
ATCC 43078



Streptococcus equi subsp. equi
ATCC 9528



Streptococcus ferus
ATCC 33477



Streptococcus gordonii
ATCC 10558



Streptococcus macacae
ATCC 35911



Streptococcus mitis
ATCC 49456



Streptococcus mutans
ATCC 25175



Streptococcus oralis
ATCC 35037



Streptococcus parasanguinis
ATCC 15912



Streptococcus parauberis
DSM 6631



Streptococcus pneumoniae
ATCC 27336



Streptococcus pyogenes
ATCC 19615



Streptococcus ratti
ATCC 19645



Streptococcus salivarius
ATCC 7073



Streptococcus sanguinis
ATCC 10556



Streptococcus sobrinus
ATCC 27352



Streptococcus suis
ATCC 43765



Streptococcus uberis
ATCC 19436



Streptococcus vestibularis
ATCC 49124

















TABLE 10







Bacterial species used to test the specificity of the Enterococcus-


specific amplification primers derived from tuf sequences.










Strain
Reference number











Gram-positive species (n = 74)










Abiotrophia adiacens
ATCC 49176



Abiotrophia defectiva
ATCC 49175



Bacillus cereus
ATCC 14579



Bacillus subtilis
ATCC 27370



Bifidobacterium adolescentis
ATCC 27534



Bifidobacterium breve
ATCC 15700



Bifidobacterium dentium
ATCC 27534



Bifidobacterium longum
ATCC 15707



Clostridium perfringens
ATCC 3124



Clostridium septicum
ATCC 12464



Corynebacterium aquaticus
ATCC 14665



Corynebacterium
ATCC 10700



pseudodiphtheriticum



Enterococcus avium
ATCC 14025



Enterococcus casseliflavus
ATCC 25788



Enterococcus cecorum
ATCC 43199



Enterococcus columbae
ATCC 51263



Enterococcus dispar
ATCC 51266



Enterococcus durans
ATCC 19432



Enterococcus faecalis
ATCC 29212



Enterococcus faecium
ATCC 19434



Enterococcus flavescens
ATCC 49996



Enterococcus gallinarum
ATCC 49573



Enterococcus hirae
ATCC 8044



Enterococcus malodoratus
ATCC 43197



Enterococcus mundtii
ATCC 43186



Enterococcus pseudoavium
ATCC 49372



Enterococcus raffinosus
ATCC 49427



Enterococcus saccharolyticus
ATCC 43076



Enterococcus solitarius
ATCC 49428



Enterococcus sulfureus
ATCC 49903



Eubacterium lentum
ATCC 49903



Gemella haemolysans
ATCC 10379



Gemella morbillorum
ATCC 27842



Lactobacillus acidophilus
ATCC 4356



Leuconostoc mesenteroides
ATCC 19225



Listeria grayi
ATCC 19120



Listeria grayi
ATCC 19123



Listeria innocua
ATCC 33090



Listeria ivanovii
ATCC 19119



Listeria monocytogenes
ATCC 15313



Listeria seeligeri
ATCC 35967



Micrococcus luteus
ATCC 9341



Pediococcus acidilacti
ATCC 33314



Pediococcus pentosaceus
ATCC 33316



Peptococcus niger
ATCC 27731



Peptostreptococcus anaerobius
ATCC 27337



Peptostreptococcus indolicus
ATCC 29247



Peptostreptococcus micros
ATCC 33270



Propionibacterium acnes
ATCC 6919



Staphylococcus aureus
ATCC 43300



Staphylococcus capitis
ATCC 27840



Staphylococcus epidermidis
ATCC 14990



Staphylococcus haemolyticus
ATCC 29970



Staphylococcus hominis
ATCC 27844



Staphylococcus lugdunensis
ATCC 43809



Staphylococcus saprophyticus
ATCC 15305



Staphylococcus simulans
ATCC 27848



Staphylococcus warneri
ATCC 27836



Streptococcus agalactiae
ATCC 13813



Streptococcus anginosus
ATCC 33397



Streptococcus bovis
ATCC 33317



Streptococcus constellatus
ATCC 27823



Streptococcus cristatus
ATCC 51100



Streptococcus intermedius
ATCC 27335



Streptococcus mitis
ATCC 49456



Streptococcus mitis
ATCC 3639



Streptococcus mutans
ATCC 27175



Streptococcus parasanguinis
ATCC 15912



Streptococcus pneumoniae
ATCC 27736



Streptococcus pneumoniae
ATCC 6303



Streptococcus pyogenes
ATCC 19615



Streptococcus salivarius
ATCC 7073



Streptococcus sanguinis
ATCC 10556



Streptococcus suis
ATCC 43765







Gram-negative species (n = 39)










Acidominococcus fermentans
ATCC 2508



Acinetobacter baumannii
ATCC 19606



Alcaligenes faecalis
ATCC 8750



Anaerobiospirillum
ATCC 29305



succiniproducens



Anaerorhabdus furcosus
ATCC 25662



Bacteroides distasonis
ATCC 8503



Bacteroides thetaiotaomicron
ATCC 29741



Bacteroides vulgatus
ATCC 8482



Bordetella pertussis
LSPQ 3702



Bulkholderia cepacia
LSPQ 2217



Butyvibrio fibrinosolvens
ATCC 19171



Cardiobacterium hominis
ATCC 15826



Citrobacter freundii
ATCC 8090



Desulfovibrio vulgaris
ATCC 29579



Edwardsiellae tarda
ATCC 15947



Enterobacter cloacae
ATCC 13047



Escherichia coli
ATCC 25922



Fusobacterium russii
ATCC 25533



Haemophilus influenzae
ATCC 9007



Hafnia alvei
ATCC 13337



Klebsiella oxytoca
ATCC 13182



Meganomonas hypermegas
ATCC 25560



Mitsukoella multiacidus
ATCC 27723



Moraxella catarrhalis
ATCC 43628



Morganella morganii
ATCC 25830



Neisseria meningitidis
ATCC 13077



Pasteurella aerogenes
ATCC 27883



Proteus vulgaris
ATCC 13315



Providencia alcalifaciens
ATCC 9886



Providencia rettgeri
ATCC 9250



Pseudomonas aeruginosa
ATCC 27853



Salmonella typhimurium
ATCC 14028



Serratia marcescens
ATCC 13880



Shigella flexneri
ATCC 12022



Shigella sonnei
ATCC 29930



Succinivibrio dextrinosolvens
ATCC 19716



Tissierella praeacuta
ATCC 25539



Veillonella parvuala
ATCC 10790



Yersinia enterocolitica
ATCC 9610

















TABLE 11







Microbial species for which tuf and/or atpD and/or recA sequences are available in public


databases.










Species
Strain
Accession number
Coding gene*










tuf sequences










Bacteria





Actinobacillus actinomycetemcomitans
HK1651
Genome project2
tuf


Actinobacillus actinomycetemcomitans
HK1651
Genome project2
tuf (EF-G)


Agrobacterium tumefaciens

X99673
tuf


Agrobacterium tumefaciens

X99673
tuf (EF-G)


Agrobacterium tumefaciens

X99674
tuf


Anacystis nidulans
PCC 6301
X17442
tuf


Aquifex aeolicus
VF5
AE000669
tuf


Aquifex aeolicus
VF5
AE000669
tuf (EF-G)


Aquifex pyrophilus

Genome project2
tuf (EF-G)


Aquifex pyrophilus

Y15787
tuf


Bacillus anthracis
Ames
Genome project2
tuf


Bacillus anthracis
Ames
Genome project2
tuf (EF-G)


Bacillus halodurans
C-125
AB017508
tuf


Bacillus halodurans
C-125
AB017508
tuf (EF-G)


Bacillus stearothermophilus
CCM 2184
AJ000260
tuf


Bacillus subtilis
168
D64127
tuf


Bacillus subtilis
168
D64127
tuf (EF-G)


Bacillus subtilis
DSM 10
Z99104
tuf


Bacillus subtilis
DSM 10
Z99104
tuf (EF-G)


Bacteroides forsythus
ATCC 43037
AB035466
tuf


Bacteroides fragilis
DSM 1151
—1
tuf


Bordetella bronchiseptica
RB50
Genome project2
tuf


Bordetella pertussis
Tohama 1
Genome project2
tuf


Bordetella pertussis
Tohama 1
Genome project2
tuf (EF-G)


Borrelia burdorgferi
B31
U78193
tuf


Borrelia burgdorferi

AE001155
tuf (EF-G)


Brevibacterium linens
DSM 20425
X76863
tuf


Buchnera aphidicola
Ap
Y12307
tuf


Burkholderia pseudomallei
K96243
Genome project2
tuf (EF-G)


Campylobacter jejuni
NCTC 11168
Y17167
tuf


Campylobacter jejuni
NCTC 11168
CJ11168X2
tuf (EF-G)


Chlamydia pneumoniae
CWL029
AE001592
tuf


Chlamydia pneumoniae
CWL029
AE001639
tuf (EF-G)


Chlamydia trachomatis

M74221
tuf


Chlamydia trachomatis
D/UW-3/CX
AE001317
tuf (EF-G)


Chlamydia trachomatis
D/UW-3/CX
AE001305
tuf


Chlamydia trachomatis
F/IC-Cal-13
L22216
tuf


Chlorobium vibrioforme
DSM 263
X77033
tuf


Chloroflexus aurantiacus
DSM 636
X76865
tuf


Clostridium acetobutylicum
ATCC 824
Genome project2
tuf


Clostridium difficile
630
Genome project2
tuf


Clostridium difficile
630
Genome project2
tuf (EF-G)


Corynebacterium diphtheriae
NCTC 13129
Genome project2
tuf


Corynebacterium diphtheriae
NCTC 13129
Genome project2
tuf (EF-G)


Corynebacterium glutamicum
ASO 19
X77034
tuf


Corynebacterium glutamicum
MJ-233
E09634
tuf


Coxiella burnetii
Nine Mile phase I
AF136604
tuf


Cytophaga lytica
DSM 2039
X77035
tuf


Deinococcus radiodurans
R1
AE001891
tuf (EF-G)


Deinococcus radiodurans
R1
AE180092
tuf


Deinococcus radiodurans
R1
AE002041
tuf


Deinonema sp.

—1
tuf


Eikenella corrodens
ATCC 23834
Z12610
tuf


Eikenella corrodens
ATCC 23834
Z12610
tuf (EF-G)


Enterococcus faecalis

Genome project2
tuf (EF-G)


Escherichia coli

J01690
tuf


Escherichia coli

J01717
tuf


Escherichia coli

X00415
tuf (EF-G)


Escherichia coli

X57091
tuf


Escherichia coli
K-12 MG1655
U00006
tuf


Escherichia coli
K-12 MG1655
U00096
tuf


Escherichia coli
K-12 MG1655
AE000410
tuf (EF-G)


Fervidobacterium islandicum
DSM 5733
Y15788
tuf


Fibrobacter succinogenes
S85
X76866
tuf


Flavobacterium ferrigeneum
DSM 13524
X76867
tuf


Flexistipes sinusarabici

X59461
tuf


Gloeobacter violaceus
PCC 7421
U09433
tuf


Gloeothece sp.
PCC 6501
U09434
tuf


Haemophilus actinomycetemcomitans
HK1651
Genome project2
tuf


Haemophilus ducreyi
35000
AF087414
tuf (EF-G)


Haemophilus influenzae
Rd
U32739
tuf


Haemophilus influenzae
Rd
U32746
tuf


Haemophilus influenzae
Rd
U32739
tuf (EF-G)


Helicobacter pylori
26695
AE000511
tuf


Helicobacter pylori
J99
AE001539
tuf (EF-G)


Helicobacter pylori
J99
AE001541
tuf


Herpetosiphon aurantiacus
Hpga1
X76868
tuf


Klebsiella pneumoniae
M6H 78578
Genome project2
tuf


Klebsiella pneumoniae
M6H 78578
Genome project2
tuf (EF-G)


Lactobacillus paracasei

E13922
tuf


Legionella pneumophila
Philadelphia-1
Genome project2
tuf


Leptospira interrogans

AF115283
tuf


Leptospira interrogans

AF115283
tuf (EF-G)


Micrococcus luteus
IFO 3333
M17788
tuf (EF-G)


Micrococcus luteus
IFO 3333
M17788
tuf


Moraxella sp.
TAC II 25
AJ249258
tuf


Mycobacterium avium
104
Genome project2
tuf


Mycobacterium avium
104
Genome project2
tuf (EF-G)


Mycobacterium bovis
AF2122/97
Genome project2
tuf


Mycobacterium bovis
AF2122/97
Genome project2
tuf (EF-G)


Mycobacterium leprae

L13276
tuf


Mycobacterium leprae

Z14314
tuf


Mycobacterium leprae

Z14314
tuf (EF-G)


Mycobacterium leprae
Thai 53
D13869
tuf


Mycobacterium tuberculosis
Erdmann
S40925
tuf


Mycobacterium tuberculosis
H37Rv
AL021943
tuf (EF-G)


Mycobacterium tuberculosis
H37Rv
Z84395
tuf


Mycobacterium tuberculosis
y42
AD000005
tuf


Mycobacterium tuberculosis
CSU#93
Genome project2
tuf


Mycobacterium tuberculosis
CSU#93
Genome project2
tuf (EF-G)


Mycoplasma capricolum
PG-31
X16462
tuf


Mycoplasma genitalium
G37
U39732
tuf


Mycoplasma genitalium
G37
U39689
tuf (EF-G)


Mycoplasma hominis

X57136
tuf


Mycoplasma hominis
PG21
M57675
tuf


Mycoplasma pneumoniae
M129
AE000019
tuf


Mycoplasma pneumoniae
M129
AE000058
tuf (EF-G)


Neisseria gonorrhoeae
MS11
L36380
tuf


Neisseria gonorrhoeae
MS11
L36380
tuf (EF-G)


Neisseria meningitidis
Z2491
Genome project2
tuf (EF-G)


Neisseria meningitidis
Z2491
Genome project2
tuf


Pasteurella multocida
Pm70
Genome project2
tuf


Peptococcus niger
DSM 20745
X76869
tuf


Phormidium ectocarpi
PCC 7375
U09443
tuf


Planobispora rosea
ATCC 53773
U67308
tuf


Planobispora rosea
ATCC 53733
X98830
tuf


Planobispora rosea
ATCC 53733
X98830
tuf (EF-G)


Plectonema boryanum
PCC 73110
U09444
tuf


Porphyromonas gingivalis
W83
Genome project2
tuf


Porphyromonas gingivalis
W83
Genome project2
tuf (EF-G)


Porphyromonas gingivalis
FDC 381
AB035461
tuf


Porphyromonas gingivalis
W83
AB035462
tuf


Porphyromonas gingivalis
SUNY 1021
AB035463
tuf


Porphyromonas gingivalis
A7A1-28
AB035464
tuf


Porphyromonas gingivalis
ATCC 33277
AB035465
tuf


Porphyromonas gingivalis
ATCC 33277
AB035471
tuf (EF-G)


Prochlorothrix hollandica

U09445
tuf


Pseudomonas aeruginosa
PAO-1
Genome project2
tuf


Pseudomonas putida

Genome project2
tuf


Rickettsia prowazekii
Madrid E
AJ235272
tuf


Rickettsia prowazekii
Madrid E
AJ235270
tuf (EF-G)


Rickettsia prowazekii
Madrid E
Z54171
tuf (EF-G)


Salmonella choleraesuis subsp.

X64591
tuf (EF-G)


choleraesuis serotype Typhimurium


Salmonella choleraesuis subsp.
LT2 trpE91
X55116
tuf


choleraesuis serotype Typhimurium


Salmonella choleraesuis subsp.
LT2 trpE91
X55117
tuf


choleraesuis serotype Typhimurium


Serpulina hyodysenteriae
B204
U51635
tuf


Serratia marcescens

AF058451
tuf


Shewanella putrefaciens
DSM 50426
—1
tuf


Shewanella putrefaciens
MR-1
Genome project2
tuf


Spirochaeta aurantia
DSM 1902
X76874
tuf


Staphylococcus aureus

AJ237696
tuf (EF-G)


Staphylococcus aureus
EMRSA-16
Genome project2
tuf


Staphylococcus aureus
NCTC 8325
Genome project2
tuf


Staphylococcus aureus
COL
Genome project2
tuf


Staphylococcus aureus
EMRSA-16
Genome project2
tuf (EF-G)


Stigmatella aurantiaca
DW4
X82820
tuf


Stigmatella aurantiaca
Sg a1
X76870
tuf


Streptococcus mutans
GS-5 Kuramitsu
U75481
tuf


Streptococcus mutans
UAB159
Genome project2
tuf


Streptococcus oralis
NTCC 11427
P331701
tuf


Streptococcus pyogenes

Genome project2
tuf (EF-G)


Streptococcus pyogenes
M1-GAS
Genome project2
tuf


Streptomyces aureofaciens
ATCC 10762
AF007125
tuf


Streptomyces cinnamoneus
Tue89
X98831
tuf


Streptomyces coelicolor
A3(2)
AL031013
tuf (EF-G)


Streptomyces coelicolor
A3(2)
X77039
tuf (EF-G)


Streptomyces coelicolor
M145
X77039
tuf


Streptomyces collinus
BSM 40733
S79408
tuf


Streptomyces netropsis
Tu1063
AF153618
tuf


Streptomyces ramocissimus

X67057
tuf


Streptomyces ramocissimus

X67058
tuf


Streptomyces ramocissimus

X67057
tuf (EF-G)


Synechococcus sp.
PCC 6301
X17442
tuf (EF-G)


Synechococcus sp.
PCC 6301
X17442
tuf


Synechocystis sp.
PCC 6803
D90913
tuf (EF-G)


Synechocystis sp.
PCC 6803
D90913
tuf


Synechocystis sp.
PCC 6803
X65159
tuf (EF-G)


Taxeobacter occealus
Myx 2105
X77036
tuf


Thermotoga maritima

Genome project2
tuf (EF-G)


Thermotoga maritima

M27479
tuf


Thermus aquaticus
EP 00276
X66322
tuf


Thermus thermophilus
HB8
X16278
tuf (EF-G)


Thermus thermophilus
HB8
X05977
tuf


Thermus thermophilus
HB8
X06657
tuf


Thiomonas cuprina
DSM 5495
U78300
tuf


Thiomonas cuprina
DSM 5495
U78300
tuf (EF-G)


Thiomonas cuprina
Hoe5
X76871
tuf


Treponema denticola

Genome project2
tuf


Treponema denticola

Genome project2
tuf (EF-G)


Treponema pallidum

AE001202
tuf


Treponema pallidum

AE001222
tuf (EF-G)


Treponema pallidum

AE001248
tuf (EF-G)


Ureaplasma urealyticum
ATCC 33697
Z34275
tuf


Ureaplasma urealyticum
serovar 3 biovar 1
AE002151
tuf


Ureaplasma urealyticum
serovar 3 biovar 1
AE002151
tuf (EF-G)


Vibrio cholerae
N16961
Genome project2
tuf


Wolinella succinogenes
DSM 1740
X76872
tuf


Yersinia pestis
CO-92
Genome project2
tuf


Yersinia pestis
CO-92
Genome project2
tuf (EF-G)


Archaebacteria


Archaeoglobus fulgidus

Genome project2
tuf (EF-G)


Halobacterium marismortui

X16677
tuf


Methanobacterium thermoautrophicum
delta H
AE000877
tuf


Methanococcus jannaschii
ATCC 43067
U67486
tuf


Methanococcus vannielii

X05698
tuf


Pyrococcus abyssi
Orsay
AJ248285
tuf


Thermoplasma acidophilum
DSM 1728
X53866
tuf


Fungi


Absidia glauca
CBS 101.48
X54730
tuf (EF-1)


Arxula adeninivorans
Ls3
Z47379
tuf (EF-1)


Aspergillus oryzae
KBN616
AB007770
tuf (EF-1)


Aureobasidium pullulans
R106
U19723
tuf (EF-1)


Candida albicans
SC5314
Genome project2
tuf (M)


Candida albicans
SC5314
M29934
tuf (EF-1)


Candida albicans
SC5314
M29935
tuf (EF-1)


Cryptococcus neoformans
B3501
U81803
tuf (EF-1)


Cryptococcus neoformans
M1-106
U81804
tuf (EF-1)


Eremothecium gossypii
ATCC 10895
X73978
tuf (EF-1)


Eremothecium gossypii

A29820
tuf (EF-1)


Fusarium oxysporum
NRRL 26037
AF008498
tuf (EF-1)


Histoplasma capsulatum
186AS
U14100
tuf (EF-1)


Podospora anserina

X74799
tuf (EF-1)


Podospora curvicolla
VLV
X96614
tuf (EF-1)


Prototheca wickerhamii
263-11
AJ245645
tuf (EF-1)


Puccinia graminis
race 32
X73529
tuf (EF-1)


Reclinomonas americana
ATCC 50394
AF007261
tuf (M)


Rhizomucor racemosus
ATCC 1216B
X17475
tuf (EF-1)


Rhizomucor racemosus
ATCC 1216B
J02605
tuf (EF-1)


Rhizomucor racemosus
ATCC 1216B
X17476
tuf (EF-1)


Rhodotorula mucilaginosa

AF016239
tuf (EF-1)


Saccharomyces cerevisiae

K00428
tuf (M)


Saccharomyces cerevisiae

M59369
tuf (EF-G)


Saccharomyces cerevisiae

X00779
tuf (EF-1)


Saccharomyces cerevisiae

X01638
tuf (EF-1)


Saccharomyces cerevisiae

M10992
tuf (EF-1)


Saccharomyces cerevisiae
Alpha S288
X78993
tuf (EF-1)


Saccharomyces cerevisiae

M15666
tuf (EF-1)


Saccharomyces cerevisiae

Z35987
tuf (EF-1)


Saccharomyces cerevisiae
S288C (AB972)
U51033
tuf (EF-1)


Schizophyllum commune
1-40
X94913
tuf (EF-1)


Schizosaccharomyces pombe
972h-
AL021816
tuf (EF-1)


Schizosaccharomyces pombe
972h-
AL021813
tuf (EF-1)


Schizosaccharomyces pombe
972h-
D82571
tuf (EF-1)


Schizosaccharomyces pombe

U42189
tuf (EF-1)


Schizosaccharomyces pombe
PR745
D89112
tuf (EF-1)


Sordaria macrospora
OOO
X96615
tuf (EF-1)


Trichoderma reesei
QM9414
Z23012
tuf (EF-1)


Yarrowia lipolytica

AF054510
tuf (EF-1)


Parasites


Blastocystis hominis
HE87-1
D64080
tuf (EF-1)


Cryptosporidium parvum

U69697
tuf (EF-1)


Eimeria tenella
LS18
AI755521
tuf (EF-1)


Entamoeba histolytica
HM1:IMSS
X83565
tuf (EF-1)


Entamoeba histolytica
NIH 200
M92073
tuf (EF-1)


Giardia lamblia

D14342
tuf (EF-1)


Kentrophoros sp.

AF056101
tuf (EF-1)


Leishmania amazonensis
IFLA/BR/67/PH8
M92653
tuf (EF-1)


Leishmania braziliensis

U72244
tuf (EF-1)


Onchocerca volvulus

M64333
tuf (EF-1)


Porphyra purpurea
Avonport
U08844
tuf (EF-1)


Plasmodium berghei
ANKA
AJ224150
tuf (EF-1)


Plasmodium falciparum
K1
X60488
tuf (EF-1)


Plasmodium knowlesi
line H
AJ224153
tuf (EF-1)


Toxoplasma gondii
RH
Y11431
tuf (EF-1)


Trichomonas tenax
ATCC 30207
D78479
tuf (EF-1)


Trypanosoma brucei
LVH/75/USAMRU-K/18Y
U10562
tuf (EF-1)


Trypanosoma cruzi

L76077
tuf (EF-1)


Human and plants


Arabidopsis thaliana
Columbia
X89227
tuf (EF-1)


Glycine max
Ceresia
X89058
tuf (EF-1)


Glycine max
Ceresia
Y15107
tuf (EF-1)


Glycine max
Ceresia
Y15108
tuf (EF-1)


Glycine max
Maple Arrow
X66062
tuf (EF-1)


Homo sapiens

X03558
tuf (EF-1)


Pyramimonas disomata

AB008010
tuf







atpD sequences










Bacteria





Acetobacterium woodi
DSM 1030
U10505
atpD


Actinobacillus actinomycetemcomitans
HK1651
Genome project2
atpD


Bacillus anthracis
Ames
Genome project2
atpD


Bacillus firmus
OF4
M60117
atpD


Bacillus megaterium
QM B1551
M20255
atpD


Bacillus stearothermophilus

D38058
atpD


Bacillus stearothermophilus
IFO1035
D38060
atpD


Bacillus subtilis
168
Z28592
atpD


Bacteroides fragilis
DSM 2151
M22247
atpD


Bordetella bronchiseptica
RB50
Genome project2
atpD


Bordetella pertussis
Tohama 1
Genome project2
atpD


Borrelia burgdorferi
B31
AE001122
atpD (V)


Burkholderia cepacia
DSM50181
X76877
atpD


Burkholderia pseudomallei
K96243
Genome project2
atpD


Campylobacter jejuni
NCTC 11168
CJ11168X1
atpD


Chlamydia pneumoniae

Genome project2
atpD (V)


Chlamydia trachomatis
MoPn
Genome project2
atpD (V)


Chlorobium vibrioforme
DSM 263
X76873
atpD


Citrobacter freundii
JEO503
AF037156
atpD


Clostridium acetobutylicum
ATCC 824
Genome project2
atpD


Clostridium acetobutylicum
DSM 792
AF101055
atpD


Clostridium difficile
630
Genome project2
atpD


Corynebacterium diphtheriae
NCTC13129
Genome project2
atpD


Corynebacterium glutamicum
ASO 19
X76875
atpD


Corynebacterium glutamicum
MJ-233
E09634
atpD


Cytophaga lytica
DSM 2039
M22535
atpD


Enterobacter aerogenes
DSM 30053
—3
atpD


Enterococcus faecalis
V583
Genome project2
atpD (V)


Enterococcus hirae

M90060
atpD


Enterococcus hirae
ATCC 9790
D17462
atpD (V)


Escherichia coli

J01594
atpD


Escherichia coli

M25464
atpD


Escherichia coli

V00267
atpD


Escherichia coli

V00311
atpD


Escherichia coli
K12 MG1655
L10328
atpD


Flavobacterium ferrugineum
DSM 13524
—3
atpD


Haemophilus actinomycetemcomitans

Genome project2
atpD


Haemophilus influenzae
Rd
U32730
atpD


Helicobacter pylori
NCTC 11638
AF004014
atpD


Helicobacter pylori
26695
Genome project2
atpD


Helicobacter pylori
J99
Genome project2
atpD


Klebsiella pneumoniae
M6H 78578
Genome project2
atpD


Lactobacillus casei
DSM 20021
X64542
atpD


Legionella pneumophila
Philadelphia-1
Genome project2
atpD


Moorella thermoacetica
ATCC 39073
U64318
atpD


Mycobacterium avium
104
Genome project2
atpD


Mycobacterium bovis
AF2122/97
Genome project2
atpD


Mycobacterium leprae

U15186
atpD


Mycobacterium leprae

Genome project2
atpD


Mycobacterium tuberculosis
H37Rv
Z73419
atpD


Mycobacterium tuberculosis
CSU#93
Genome project2
atpD


Mycoplasma gallisepticum

X64256
atpD


Mycoplasma genitalium
G37
U39725
atpD


Mycoplasma pneumoniae
M129
U43738
atpD


Neisseria gonorrhoeae
FA 1090
Genome project2
atpD


Neisseria meningitidis
Z2491
Genome project2
atpD


Pasteurella multocida
Pm70
Genome project2
atpD


Pectinatus frisingensis
DSM 20465
X64543
atpD


Peptococcus niger
DSM 20475
X76878
atpD


Pirellula marina
IFAM 1313
X57204
atpD


Porphyromonas gingivalis
W83
Genome project2
atpD (V)


Propionigenium modestum
DSM 2376
X58461
atpD


Pseudomonas aeruginosa
PAO1
Genome project2
atpD


Pseudomonas putida

Genome project2
atpD


Rhodobacter capsulatus
B100
X99599
atpD


Rhodospirillum rubrum

X02499
atpD


Rickettsia prowazekii
F-12
AF036246
atpD


Rickettsia prowazekii
Madrid
Genome project2
atpD


Ruminococcus albus
7ATCC
AB006151
atpD


Salmonella bongori
JEO4162
AF037155
atpD


Salmonella bongori
BR1859
AF037154
atpD


Salmonella choleraesuis
S83769
AF037146
atpD


subsp. arizonae


Salmonella choleraesuis
u24
AF037147
atpD


subsp. arizonae


Salmonella choleraesuis subsp.
K228
AF037140
atpD


choleraesuis serotype Dublin


Salmonella choleraesuis subsp.
K771
AF037139
atpD


choleraesuis serotype Dublin


Salmonella choleraesuis subsp.
Div36-86
AF037142
atpD


choleraesuis serotype Infantis


Salmonella choleraesuis subsp.
Div95-86
AF037143
atpD


choleraesuis serotype Tennessee


Salmonella choleraesuis subsp.
LT2
AF037141
atpD


choleraesuis serotype Typhimurium


Salmonella choleraesuis
DS210/89
AF037149
atpD


subsp. diarizonae


Salmonella choleraesuis
JEO307
AF037148
atpD


subsp. diarizonae


Salmonella choleraesuis
S109671
AF037150
atpD


subsp. diarizonae


Salmonella choleraesuis
S84366
AF037151
atpD


subsp. houtenae


Salmonella choleraesuis
S84098
AF037152
atpD


subsp. houtenae


Salmonella choleraesuis
BR2047
AF037153
atpD


subsp. indica


Salmonella choleraesuis
NSC72
AF037144
atpD


subsp. salamae


Salmonella choleraesuis
S114655
AF037145
atpD


subsp. salamae


Shewanella putrefaciens
MR-1
Genome project2
atpD


Staphylococcus aureus
COL
Genome project2
atpD


Stigmatella aurantiaca
Sga1
X76879
atpD


Streptococcus bovis
JB-1
AB009314
atpD


Streptococcus mutans
GS-5
U31170
atpD


Streptococcus mutans
UAB159
Genome project2
atpD


Streptococcus pneumoniae
Type 4
Genome project2
atpD (V)


Streptococcus pneumoniae
Type 4
Genome project2
atpD


Streptococcus pyogenes
M1-GAS
Genome project2
atpD (V)


Streptococcus pyogenes
M1-GAS
Genome project2
atpD


Streptococcus sanguinis
10904
AF001955
atpD


Streptomyces lividans
1326
Z22606
atpD


Thermus thermophilus
HB8
D63799
atpD (V)


Thiobacillus ferrooxidans
ATCC 33020
M81087
atpD


Treponema pallidum
Nichols
AE001228
atpD (V)


Vibrio alginolyticus

X16050
atpD


Vibrio cholerae
N16961
Genome project2
atpD


Wolinella succinogenes
DSM 1470
X76880
atpD


Yersinia enterocolitica
NCTC 10460
AF037157
atpD


Yersinia pestis
CO-92
Genome project2
atpD


Archaebacteria


Archaeoglobus fulgidus
DSM 4304
AE001023
atpD (V)


Halobacterium salinarum

S56356
atpD (V)


Haloferax volcanii
WR 340
X79516
atpD


Methanococcus jannaschii
DSM 2661
U67477
atpD (V)


Methanosarcina barkeri
DSM 800
J04836
atpD (V)


Fungi


Candida albicans
SC5314
Genome project2
atpD


Candida tropicalis

M64984
atpD (V)


Kluyveromyces lactis
2359/152
U37764
atpD


Neurospora crassa

X53720
atpD


Saccharomyces cerevisiae

M12082
atpD


Saccharomyces cerevisiae
X2180-1A
J05409
atpD (V)


Schizosaccharomyces pombe
972 h-
S47814
atpD (V)


Schizosaccharomyces pombe
972 h-
M57956
atpD


Parasites


Giardia lamblia
WB
U18938
atpD


Plasmodium falciparum
3D7
L08200
atpD (V)


Trypanosoma congolense
IL3000
Z25814
atpD (V)


Human and plants


Homo sapiens

L09234
atpD (V)


Homo sapiens

M27132
atpD







recA sequences










Bacteria





Acetobacter aceti
no. 1023
S60630
recA


Acetobacter altoacetigenes
MH-24
E05290
recA


Acetobacter polyoxogenes
NBI 1028
D13183
recA


Acholeplasma laidlawii
8195
M81465
recA


Acidiphilium facilis
ATCC 35904
D16538
recA


Acidothermus cellulolyticus
ATCC 43068
AJ006705
recA


Acinetobacter calcoaceticus
BD413/ADP1
L26100
recA


Actinobacillus actinomycetemcomitans
HK1651
Genome project2
recA


Aeromonas salmonicida
A449
U83688
recA


Agrobacterium tumefaciens
C58
L07902
recA


Allochromatium vinosum

AJ000677
recA


Aquifex aeolicus
VF5
AE000775
recA


Aquifex pyrophilus
Kol5a
L23135
recA


Azotobacter vinelandii

S96898
recA


Bacillus stearothermophilus
10
Genome project2
recA


Bacillus subtilis
PB1831
U87792
recA


Bacillus subtilis
168
Z99112
recA


Bacteroides fragilis

M63029
recA


Bifidobacterium breve
NCFB 2258
AF094756
recA


Blastochloris viridis
DSM 133
AF022175
recA


Bordetella pertussis
165
X53457
recA


Bordetella pertussis
Tohama I
Genome project2
recA


Borrelia burgdorferi
Sh-2-82
U23457
recA


Borrelia burgdorferi
B31
AE001124
recA


Brevibacterium flavum
MJ-233
E10390
recA


Brucella abortus
2308
L00679
recA


Burkholderia cepacia
ATCC 17616
U70431
recA


Burkholderia cepacia

D90120
recA


Burkholderia pseudomallei
K96243
Genome project2
recA


Campylobacter fetus subsp. fetus
23D
AF020677
recA


Campylobacter jejuni
81-176
U03121
recA


Campylobacter jejuni
NCTC 11168
AL139079
recA


Chlamydia trachomatis
L2
U16739
recA


Chlamydia trachomatis
D/UW-3/CX
AE001335
recA


Chlamydophila pneumoniae
CWL029
AE001658
recA


Chloroflexus aurantiacus
J-10-fl
AF037259
recA


Clostridium acetobutylicum

M94057
recA


Clostridium perfringens
13
U61497
recA


Corynebacterium diphtheriae
NCTC13129
Genome project2
recA


Corynebacterium glutamicum
AS019
U14965
recA


Corynebacterium pseudotuberculosis
C231
U30387
recA


Deinococcus radiodurans
KD8301
AB005471
recA


Deinococcus radiodurans
R1
U01876
recA


Enterobacter agglomerans
339
L03291
recA


Enterococcus faecalis
OGIX
M81466
recA


Erwinia carotovora

X55554
recA


Escherichia coli

J01672
recA


Escherichia coli

X55552
recA


Escherichia coli
K-12
AE000354
recA


Frankia alni
Arl3
AJ006707
recA


Gluconobacter oxydans

U21001
recA


Haemophilus influenzae
Rd
U32687
recA


Haemophilus influenzae
Rd
U32741
recA


Haemophilus influenzae
Rd
L07529
recA


Helicobacter pylori
69A
Z35478
recA


Helicobacter pylori
26695
AE000536
recA


Helicobacter pylori
J99
AE001453
recA


Klebsiella pneumoniae
M6H 78578
Genome project2
recA


Lactococcus lactis
ML3
M88106
recA


Legionella pneumophila

X55453
recA


Leptospira biflexa
serovar patoc
U32625
recA


Leptospira interrogans
serovar pomona
U29169
recA


Magnetospirillum magnetotacticum
MS-1
X17371
recA


Methylobacillus flagellatus
MFK1
M35325
recA


Methylomonas clara
ATCC 31226
X59514
recA


Mycobacterium avium
104
Genome project2
recA


Mycobacterium bovis
AF122/97
Genome project2
recA


Mycobacterium leprae

X73822
recA


Mycobacterium tuberculosis
H37Rv
X58485
recA


Mycobacterium tuberculosis
CSU#93
Genome project2
recA


Mycoplasma genitalium
G37
U39717
recA


Mycoplasma mycoides
GM9
L22073
recA


Mycoplasma pneumoniae
ATCC 29342
MPAE000033
recA


Mycoplasma pulmonis
KD735
L22074
recA


Myxococcus xanthus

L40368
recA


Myxococcus xanthus

L40367
recA


Neisseria animalis
NCTC 10212
U57910
recA


Neisseria cinerea
LCDC 81-176
AJ223869
recA


Neisseria cinerea
LNP 1646
U57906
recA


Neisseria cinerea
NCTC 10294
AJ223871
recA


Neisseria cinerea
Vedros M601
AJ223870
recA


Neisseria elongata
CCUG 2131
AJ223882
recA


Neisseria elongata
CCUG 4165A
AJ223880
recA


Neisseria elongata
NCTC 10660
AJ223881
recA


Neisseria elongata
NCTC 11050
AJ223878
recA


Neisseria elongata
NHITCC 2376
AJ223877
recA


Neisseria elongata
CCUG 4557
AJ223879
recA


subsp. intermedia


Neisseria flava
Bangor 9
AJ223873
recA


Neisseria flavescens
LNP 444
U57907
recA


Neisseria gonorrhoeae
CH95
U57902
recA


Neisseria gonorrhoeae
FA19
X64842
recA


Neisseria gonorrhoeae
MS11
X17374
recA


Neisseria gonorrhoeae

Genome project2
recA


Neisseria lactamica
CCUC 7757
AJ223866
recA


Neisseria lactamica
CCUG 7852
Y11819
recA


Neisseria lactamica
LCDC 77-143
Y11818
recA


Neisseria lactamica
LCDC 80-111
AJ223864
recA


Neisseria lactamica
LCDC 845
AJ223865
recA


Neisseria lactamica
NCTC 10617
U57905
recA


Neisseria lactamica
NCTC 10618
AJ223863
recA


Neisseria meningitidis
44/46
X64849
recA


Neisseria meningitidis
Bangor 13
AJ223868
recA


Neisseria meningitidis
HF116
X64848
recA


Neisseria meningitidis
HF130
X64844
recA


Neisseria meningitidis
HF46
X64847
recA


Neisseria meningitidis
M470
X64850
recA


Neisseria meningitidis
N94II
X64846
recA


Neisseria meningitidis
NCTC 8249
AJ223867
recA


Neisseria meningitidis
P63
X64845
recA


Neisseria meningitidis
S3446
U57903
recA


Neisseria meningitidis
FAM18
Genome project2
recA


Neisseria mucosa
LNP 405
U57908
recA


Neisseria mucosa
Vedros M1801
AJ223875
recA


Neisseria perflava
CCUG 17915
AJ223876
recA


Neisseria perflava
LCDC 85402
AJ223862
recA


Neisseria pharyngis var. flava
NCTC 4590
U57909
recA


Neisseria polysaccharea
CCUG 18031
Y11815
recA


Neisseria polysaccharea
CCUG 24845
Y11816
recA


Neisseria polysaccharea
CCUG 24846
Y11814
recA


Neisseria polysaccharea
INS MA 3008
Y11817
recA


Neisseria polysaccharea
NCTC 11858
U57904
recA


Neisseria sicca
NRL 30016
AJ223872
recA


Neisseria subflava
NRL 30017
AJ223874
recA


Paracoccus denitrificans
DSM 413
U59631
recA


Pasteurella multocida

X99324
recA


Porphyromonas gingivalis
W83
U70054
recA


Prevotella ruminicola
JCM 8958
U61227
recA


Proteus mirabilis
pG1300
X14870
recA


Proteus vulgaris

X55555
recA


Pseudomonas aeruginosa

X05691
recA


Pseudomonas aeruginosa
PAM 7
X52261
recA


Pseudomonas aeruginosa
PAO12
D13090
recA


Pseudomonas fluorescens
OE 28.3
M96558
recA


Pseudomonas putida

L12684
recA


Pseudomonas putida
PpS145
U70864
recA


Rhizobium leguminosarum
VF39
X59956
recA


biovar viciae


Rhizobium phaseoli
CNPAF512
X62479
recA


Rhodobacter capsulatus
J50
X82183
recA


Rhodobacter sphaeroides
2.4.1
X72705
recA


Rhodopseudomonas palustris
N 7
D84467
recA


Rickettsia prowazekii
Madrid E
AJ235273
recA


Rickettsia prowazekii
Madrid E
U01959
recA


Serratia marcescens

M22935
recA


Shigella flexneri

X55553
recA


Shigella sonnei
KNIH104S
AF101227
recA


Sinorhizobium meliloti
2011
X59957
recA


Staphylococcus aureus

L25893
recA


Streptococcus gordonii
Challis V288
L20574
recA


Streptococcus mutans
UA96
M81468
recA


Streptococcus mutans
GS-5
M61897
recA


Streptococcus pneumoniae

Z17307
recA


Streptococcus pneumoniae
R800
Z34303
recA


Streptococcus pyogenes
NZ131
U21934
recA


Streptococcus pyogenes
D471
M81469
recA


Streptococcus salivarius

M94062
recA


subsp. thermophilus


Streptomyces ambofaciens
DSM 40697
Z30324
recA


Streptomyces coelicolor
A3(2)
AL020958
recA


Streptomyces lividans
TK24
X76076
recA


Streptomyces rimosus
R6
X94233
recA


Streptomyces venezuelae
ATCC10712
U04837
recA


Synechococcus sp.
PR6
M29495
recA


Synechocystis sp.
PCC6803
D90917
recA


Thermotoga maritima

L23425
recA


Thermotoga maritima

AE001823
recA


Thermus aquaticus

L20095
recA


Thermus thermophilus
HB8
D17392
recA


Thiobacillus ferrooxidans

M26933
recA


Treponema denticola

Genome project2
recA


Treponema pallidum
Nichols
AE001243
recA


Vibrio anguillarum

M80525
recA


Vibrio cholerae
017
X71969
recA


Vibrio cholerae
2740-80
U10162
recA


Vibrio cholerae
569B
L42384
recA


Vibrio cholerae
M549
AF117881
recA


Vibrio cholerae
M553
AF117882
recA


Vibrio cholerae
M645
AF117883
recA


Vibrio cholerae
M793
AF117878
recA


Vibrio cholerae
M794
AF117880
recA


Vibrio cholerae
M967
AF117879
recA


Xanthomonas citri
XW47
AF006590
recA


Xanthomonas oryzae

AF013600
recA


Xenorhabdus bovienii
T228/1
U87924
recA


Xenorhabdus nematophilus
AN6
AF127333
recA


Yersinia pestis
231
X75336
recA


Yersinia pestis
CO-92
Genome project2
recA


Fungi, parasites, human and plants


Anabaena variabilis
ATCC 29413
M29680
recA


Arabidopsis thaliana

U43652
recA (Rad51)


Candida albicans

U39808
recA (Dmc1)


Coprinus cinereus
Okayama-7
U21905
recA (Rad51)


Emericella nidulans

Z80341
recA (Rad51)


Gallus gallus

L09655
recA (Rad51)


Homo sapiens

D13804
recA (Rad51)


Homo sapiens

D63882
recA (Dmc1)


Leishmania major
Friedlin
AF062379
recA (Rad51)


Leishmania major
Friedlin
AF062380
recA (Dmc1)


Mus musculus

D58419
recA (Dmc1)


Neurospora crassa
74-OR23-1A
D29638
recA (Rad51)


Saccharomyces cerevisiae

D10023
recA (Rad51)


Schizosaccharomyces pombe

Z22691
recA (Rad51)


Schizosaccharomyces pombe
972h-
AL021817
recA (Dmc1)


Tetrahymena thermophila
PB9R
AF064516
recA (Rad51)


Trypanosoma brucei
stock 427
Y13144
recA (Rad51)


Ustilago maydis

U62484
recA (Rad51)


Xenopus laevis

D38488
recA (Rad51)


Xenopus laevis

D38489
recA (Rad51)





*tuf indicates tuf sequences, including tuf genes, fusA genes and fusA-tuf intergenic spacers.


tuf (C) indicates tuf sequences divergent from main (usually A and B) copies of the elongation factor-Tu


tuf (EF-1) indicates tuf sequences of the eukaryotic type (elongation factor 1α)


tuf (M) indicates tuf sequences from organellar (mostly mitochondrial) origin


atpD indicates atpD sequences ot the F-type


atpD (V) indicates atpD sequences of the V-Type


recA indicates recA sequences


recA (Rad51) indicates rad51 sequences or homologs


recA (Dmc1) indicates dmc1 sequences or homologs


1Nucleotides sequences published in Arch. Microbiol. 1990 153: 241-247


2These sequences are from theTIGR database (http://www.tigr.org/tdb/tdb.html)


3Nucleotides sequences published in FEMS Microbiology Letters 1988 50: 101-106













TABLE 12







Bacterial species used to test the specificity of the Staphylococcus-


specific amplification primers derived from tuf sequences.










Strain
Reference number











Staphylococcal species (n = 27)










Staphylococcus arlettae
ATCC 43957



Staphylococcus aureus
ATCC 35844



subsp. anaerobius



Staphylococcus aureus
ATCC 43300



subsp. aureus



Staphylococcus auricularis
ATCC 33753



Staphylococcus capitis
ATCC 27840



subsp. capitis



Staphylococcus caprae
ATCC 35538



Staphylococcus carnosus
ATCC 51365



Staphylococcus chromogenes
ATCC 43764



Staphylococcus cohnii
DSM 20260



subsp. urealyticum



Staphylococcus delphini
ATCC 49171



Staphylococcus epidermidis
ATCC 14990



Staphylococcus equorum
ATCC 43958



Staphylococcus felis
ATCC 49168



Staphylococcus gallinarum
ATCC 35539



Staphylococcus haemolyticus
ATCC 29970



Staphylococcus hominis
ATCC 27844



Staphylococcus hyicus
ATCC 11249



Staphylococcus intermedius
ATCC 29663



Staphylococcus kloosis
ATCC 43959



Staphylococcus lentus
ATCC 29070



Staphylococcus lugdunensis
ATCC 43809



Staphylococcus saprophyticus
ATCC 15305



Staphylococcus schleiferi
ATCC 49545



subsp. coagulans



Staphylococcus sciuri
ATCC 29060



subsp. sciuri



Staphylococcus simulans
ATCC 27848



Staphylococcus warneri
ATCC 27836



Staphylococcus xylosus
ATCC 29971







Other Gram-positive bacteria (n = 20)










Bacillus subtilis
ATCC 27370



Enterococcus avium
ATCC 14025



Enterococcus durans
ATCC 19432



Enterococcus faecalis
ATCC 19433



Enterococcus faecium
ATCC 19434



Enterococcus flavescens
ATCC 49996



Enterococcus gallinarum
ATCC 49573



Lactobacillus acidophilus
ATCC 4356



Lactococcus lactis
ATCC 11454



Listeria innocua
ATCC 33090



Listeria ivanovii
ATCC 19119



Listeria monocytogenes
ATCC 15313



Macrococcus caseolyticus
ATCC 13548



Streptococcus agalactiae
ATCC 13813



Streptococcus anginosus
ATCC 33397



Streptococcus bovis
ATCC 33317



Streptococcus mutans
ATCC 25175



Streptococcus pneumoniae
ATCC 6303



Streptococcus pyogenes
ATCC 19615



Streptococcus salivarius
ATCC 7073







Gram-negative bacteria (n = 33)










Acinetobacter baumannii
ATCC 19606



Bacteroides distasonis
ATCC 8503



Bacteroides fragilis
ATCC 25285



Bulkholderia cepacia
ATCC 25416



Bordetella pertussis
ATCC 9797



Citrobacter freundii
ATCC 8090



Enterobacter aerogenes
ATCC 13048



Enterobacter cloacae
ATCC 13047



Escherichia coli
ATCC 25922



Haemophilus influenzae
ATCC 8907



Haemophilus parahaemolyticus
ATCC 10014



Haemophilus parainfluenzae
ATCC 7901



Hafnia alvei
ATCC 13337



Kingella indologenes
ATCC 25869



Klebsiella oxytoca
ATCC 13182



Klebsiella pneumoniae
ATCC 13883



Moraxella catarrhalis
ATCC 25240



Morganella morganii
ATCC 25830



Neisseria gonorrhoeae
ATCC 35201



Neisseria meningitidis
ATCC 13077



Proteus mirabilis
ATCC 25933



Proteus vulgaris
ATCC 13315



Providencia rettgeri
ATCC 9250



Providencia stuartii
ATCC 29914



Pseudomonas aeruginosa
ATCC 27853



Pseudomonas fluorencens
ATCC 13525



Salmonella choleraesuis
ATCC 7001



Salmonella typhimurium
ATCC 14028



Serratia marcescens
ATCC 8100



Shigella flexneri
ATCC 12022



Shigella sonnei
ATCC 29930



Stenotrophomonas maltophilia
ATCC 13843



Yersinia enterocolitica
ATCC 9610

















TABLE 13







Bacterial species used to test the specificity of the


penicillin-resistant Streptococcus pneumoniae assay.










Strain
Reference number











Gram-positive species (n = 67)










Abiotrophia adiacens
ATCC 49175



Abiotrophia defectiva
ATCC 49176



Actinomyces pyogenes
ATCC 19411



Bacillus anthracis
ATCC 4229



Bacillus cereus
ATCC 14579



Bifidobacterium breve
ATCC 15700



Clostridium difficile
ATCC 9689



Enterococcus avium
ATCC 14025



Enterococcus casseliflavus
ATCC 25788



Enterococcus dispar
ATCC 51266



Enterococcus durans
ATCC 19432



Enterococcus faecalis
ATCC 29212



Enterococcus faecium
ATCC 19434



Enterococcus flavescens
ATCC 49996



Enterococcus gallinarum
ATCC 49573



Enterococcus hirae
ATCC 8043



Enterococcus mundtii
ATCC 43186



Enterococcus raffinosus
ATCC 49427



Lactobacillus lactis
ATCC 19435



Lactobacillus monocytogenes
ATCC 15313



Mobiluncus curtisii
ATCC 35242



Peptococcus niger
ATCC 27731



Peptostreptococcus acones
ATCC 6919



Peptostreptococcus anaerobius
ATCC 27337



Peptostreptococcus
ATCC 2639



asaccharolyticus



Peptostreptococcus lactolyticus
ATCC 51172



Peptostreptococcus magnus
ATCC 15794



Peptostreptococcus prevotii
ATCC 9321



Peptostreptococcus tetradius
ATCC 35098



Staphylococcus aureus
ATCC 25923



Staphylococcus capitis
ATCC 27840



Staphylococcus epidermidis
ATCC 14990



Staphylococcus haemolyticus
ATCC 29970



Staphylococcus hominis
ATCC 27844



Staphylococcus lugdunensis
ATCC 43809



Staphylococcus saprophyticus
ATCC 15305



Staphylococcus simulans
ATCC 27848



Staphylococcus. warneri
ATCC 27836



Streptococcus acidominimus
ATCC 51726



Streptococcus agalactiae
ATCC 12403



Streptococcus anginosus
ATCC 33397



Streptococcus bovis
ATCC 33317



Streptococcus constellatus
ATCC 27823



Streptococcus cricetus
ATCC 19624



Streptococcus cristatus
ATCC 51100



Streptococcus downei
ATCC 33748



Streptococcus dysgalactiae
ATCC 43078



Streptococcus equi
ATCC 9528



Streptococcus ferus
ATCC 33477



Streptococcus gordonii
ATCC 10558



Streptococcus intermedius
ATCC 27335



Streptococcus mitis
ATCC 903



Streptococcus mitis
LSPQ 2583



Streptococcus mitis
ATCC 49456



Streptococcus mutans
ATCC 27175



Streptococcus oralis
ATCC 10557



Streptococcus oralis
ATCC 9811



Streptococcus oralis
ATCC 35037



Streptococcus parasanguinis
ATCC 15912



Streptococcus parauberis
ATCC 6631



Streptococcus rattus
ATCC 15912



Streptococcus salivarius
ATCC 7073



Streptococcus sanguinis
ATCC 10556



Streptococcus suis
ATCC 43765



Streptococcus uberis
ATCC 19436



Streptococcus vestibularis
ATCC 49124







Gram-negative species (n = 33)










Actinetobacter baumannii
ATCC 19606



Bordetella pertussis
ATCC 9797



Citrobacter diversus
ATCC 27028



Citrobacter freundii
ATCC 8090



Enterobacter aerogenes
ATCC 13048



Enterobacter agglomerans
ATCC 27155



Enterobacter cloacae
ATCC 13047



Escherichia coli
ATCC 25922



Haemophilus ducreyi
ATCC 33940



Haemophilus haemolyticus
ATCC 33390



Haemophilus influenzae
ATCC 9007



Haemophilus parainfluenzae
ATCC 7901



Hafnia alvei
ATCC 13337



Klebsiella oxytoca
ATCC 13182



Klebsiella pneumoniae
ATCC 13883



Moraxella atlantae
ATCC 29525



Moraxella catarrhalis
ATCC 43628



Moraxella morganii
ATCC 13077



Neisseria gonorrhoeae
ATCC 35201



Neisseria meningitidis
ATCC 13077



Proteus mirabilis
ATCC 25933



Proteus vulgaris
ATCC 13315



Providencia alcalifaciens
ATCC 9886



Providencia rettgeri
ATCC 9250



Providencia rustigianii
ATCC 33673



Providencia stuartii
ATCC 33672



Pseudomonas aeruginosa
ATCC 35554



Pseudomonas fluorescens
ATCC 13525



Pseudomonas stutzeri
ATCC 17588



Salmonella typhimurium
ATCC 14028



Serratia marcescens
ATCC 13880



Shigella flexneri
ATCC 12022



Yersina enterocolitica
ATCC 9610

















TABLE 14





Bacterial species (n = 104) detected by the platelet


contaminants assay. Bold characters indicate the major


bacterial contaminants found in platelet concentrates.

















Abiotrophia adiacens



Abiotrophia defectiva



Acinetobacter baumannii



Acinetobacter lwoffi



Aerococcus viridans



Bacillus anthracis



 



 



Brucella abortus



Burkholderia cepacia



Citrobacter diversus



Citrobacter freundii



Enterobacter aerogenes



Enterobacter agglomerans



 



Enterococcus avium



Enterococcus casseliflavus



Enterococcus dispar



Enterococcus durans



Enterococcus faecalis



Enterococcus faecium



Enterococcus flavescens



Enterococcus gallinarum



Enterococcus mundtii



Enterococcus raffinosus



Enterococcus solitarius



 



Gemella morbillorum



Haemophilus ducreyi



Haemophilus haemolyticus



Haemophilus influenzae



Haemophilus parahaemolyticus



Haemophilus parainfluenzae



Hafnia alvei



Kingella kingae



 



 



Legionella pneumophila



Megamonas hypermegale



Moraxella atlantae



Moraxella catarrhalis



Morganella morganii



Neisseria gonorrheae



Neisseria meningitidis



Pasteurella aerogenes



Pasteurella multocida



Peptostreptococcus magnus



Proteus mirabilis



Providencia alcalifaciens



Providencia rettgeri



Providencia rustigianii



Providencia stuartii



 



Pseudomonas fluorescens



Pseudomonas stutzeri



Salmonella bangori



 



Salmonella enteritidis



Salmonella gallinarum



Salmonella typhimurium



Serratia liquefaciens



 



Shigella flexneri



Shigella sonnei



 



Staphylococcus capitis



 



Staphylococcus haemolyticus



Staphylococcus hominis



Staphylococcus lugdunensis



Staphylococcus saprophyticus



Staphylococcus simulans



Staphylococcus warneri



Stenotrophomonas maltophilia



Streptococcus acidominimus



 



Streptococcus anginosus



Streptococcus bovis



Streptococcus constellatus



Streptococcus cricetus



Streptococcus cristatus



Streptococcus dysgalactiae



Streptococcus equi



Streptococcus ferus



Streptococcus gordonii



Streptococcus intermedius



Streptococcus macacae



Streptococcus mitis



 



Streptococcus oralis



Streptococcus parasanguinis



Streptococcus parauberis



Streptococcus pneumoniae



 



Streptococcus ratti



 



 



Streptococcus sobrinus



Streptococcus uberis



Streptococcus vestibularis



Vibrio cholerae



Yersinia enterocolitica



Yersinia pestis



 

















TABLE 15





Microorganisms identified by commercial systems1.

















Abiotrophia adiacens (Streptococcus



adjacens)



Abiotrophia defectiva (Streptococcus



defectivus)



Achromobacter species



Acidaminococcus fermentans



Acinetobacter alcaligenes



Acinetobacter anitratus



Acinetobacter baumannii



Acinetobacter calcoaceticus



Acinetobacter calcoaceticus biovar



anitratus



Acinetobacter calcoaceticus biovar



lwoffi



Acinetobacter genomo species



Acinetobacter haemolyticus



Acinetobacter johnsonii



Acinetobacter junii



Acinetobacter lwoffii



Acinetobacter radioresistens



Acinetobacter species



Actinobacillus



actinomycetemcomitans



Actinobacillus capsulatus



Actinobacillus equuli



Actinobacillus hominis



Actinobacillus lignieresii



Actinobacillus pleuropneumoniae



Actinobacillus species



Actinobacillus suis



Actinobacillus ureae



Actinomyces bovis



Actinomyces israelii



Actinomyces meyeri



Actinomyces naeslundii



Actinomyces neuii subsp. anitratus



Actinomyces neuii subsp. neuii



Actinomyces odontolyticus



Actinomyces pyogenes



Actinomyces radingae



Actinomyces species



Actinomyces turicensis



Actinomyces viscosus



Aerococcus species



Aerococcus viridans



Aeromonas caviae



Aeromonas hydrophila



Aeromonas hydrophila group



Aeromonas jandaei



Aeromonas salmonicida



Aeromonas salmonicida subsp.



achromogenes



Aeromonas salmonicida subsp.



masoucida



Aeromonas salmonicida subsp.



salmonicida



Aeromonas schubertii



Aeromonas sobria



Aeromonas species



Aeromonas trota



Aeromonas veronii



Aeromonas veronii biovar sobria



Aeromonas veronii biovar veronii



Agrobacterium radiobacter



Agrobacterium species



Agrobacterium tumefaciens



Alcaligenes denitrificans



Alcaligenes faecalis



Alcaligenes odorans



Alcaligenes odorans (Alcaligenes



faecalis)



Alcaligenes species



Alcaligenes xylosoxidans



Alcaligenes xylosoxidans subsp.



denitrificans



Alcaligenes xylosoxidans subsp.



xylosoxidans



Alloiococcus otitis



Anaerobiospirillum succiniciproducens



Anaerovibrio lipolytica



Arachnia propionica



Arcanobacterium (Actinomyces)



bemardiae



Arcanobacterium (Actinomyces)



pyogenes



Arcanobacterium haemolyticum



Arcobacter cryaerophilus



(Campylobacter cryaerophila)



Arthrobacter globiformis



Arthrobacter species



Arxiozyma telluris (Torulopsis



pintolopesii)



Atopobium minutum (Lactobacillus



minutus)



Aureobacterium species



Bacillus amyloliquefaciens



Bacillus anthracis



Bacillus badius



Bacillus cereus



Bacillus circulans



Bacillus coagulans



Bacillus firmus



Bacillus lentus



Bacillus licheniformis



Bacillus megaterium



Bacillus mycoides



Bacillus pantothenticus



Bacillus pumilus



Bacillus species



Bacillus sphaericus



Bacillus stearothermophilus



Bacillus subtilis



Bacillus thuringiensis



Bacteroides caccae



Bacteroides capillosus



Bacteroides distasonis



Bacteroides eggerthii



Bacteroides fragilis



Bacteroides merdae



Bacteroides ovatus



Bacteroides species



Bacteroides splanchnicus



Bacteroides stercoris



Bacteroides thetaiotaomicron



Bacteroides uniformis



Bacteroides ureolyticus (B. corrodens)



Bacteroides vulgatus



Bergeyella (Weeksella) zoohelcum



Bifidobacterium adolescentis



Bifidobacterium bifidum



Bifidobacterium breve



Bifidobacterium dentium



Bifidobacterium infantis



Bifidobacterium species



Blastoschizomyces (Dipodascus)



capitatus



Bordetella avium



Bordetella bronchiseptica



Bordetella parapertussis



Bordetella pertussis



Bordetella species



Borrelia species



Branhamella (Moraxella) catarrhalis



Branhamella species



Brevibacillus brevis



Brevibacillus laterosporus



Brevibacterium casei



Brevibacterium epidermidis



Brevibacterium linens



Brevibacterium species



Brevundimonas (Pseudomonas)



diminuta



Brevundimonas (Pseudomonas)



vesicularis



Brevundimonas species



Brochothrix thermosphacta



Brucella abortus



Brucella canis



Brucella melitensis



Brucella ovis



Brucella species



Brucella suis



Budvicia aquatica



Burkholderia (Pseudomonas) cepacia



Burkholderia (Pseudomonas) gladioli



Burkholderia (Pseudomonas) mallei



Burkholderia (Pseudomonas)



pseudomallei



Burkholderia species



Buttiauxella agrestis



Campylobacter coli



Campylobacter concisus



Campylobacter fetus



Campylobacter fetus subsp. fetus



Campylobacter fetus subsp.



venerealis



Campylobacter hyointestinalis



Campylobacter jejuni subsp. doylei



Campylobacter jejuni subsp. jejuni



Campylobacter lari



Campylobacter lari subsp. UPTC



Campylobacter mucosalis



Campylobacter species



Campylobacter sputorum



Campylobacter sputorum subsp.



bubulus



Campylobacter sputorum subsp.



fecalis



Campylobacter sputorum subsp.



sputorum



Campylobacter upsaliensis



Candida (Clavispora) lusitaniae



Candida (Pichia) guilliermondii



Candida (Torulopsis) glabrata



Candida albicans



Candida boidinii



Candida catenulata



Candida ciferrii



Candida colliculosa



Candida conglobata



Candida curvata (Cryptococcus



curvatus)



Candida dattila



Candida dubliniensis



Candida famata



Candida globosa



Candida hellenica



Candida holmii



Candida humicola



Candida inconspicua



Candida intermedia



Candida kefyr



Candida krusei



Candida lambica



Candida magnoliae



Candida maris



Candida melibiosica



Candida membranaefaciens



Candida norvegensis



Candida norvegica



Candida parapsilosis



Candida paratropicalis



Candida pelliculosa



Candida pseudotropicalis



Candida pulcherrima



Candida ravautii



Candida rugosa



Candida sake



Candida silvicola



Candida species



Candida sphaerica



Candida stellatoidea



Candida tenuis



Candida tropicalis



Candida utilis



Candida valida



Candida vini



Candida viswanathii



Candida zeylanoides



Capnocytophaga gingivalis



Capnocytophaga ochracea



Capnocytophaga species



Capnocytophaga sputigena



Cardiobacterium hominis



Camobacterium divergens



Camobacterium piscicola



CDC group ED-2



CDC group EF4 (Pasteurella sp.)



CDC group EF-4A



CDC group EF-4B



CDC group EQ-Z



CDC group HB-5



CDC group II K-2



CDC group IV C-2 (Bordetella-like)



CDC group M5



CDC group M6



Cedecea davisae



Cedecea lapagei



Cedecea neteri



Cedecea species



Cellulomonas (Oerskovia) turbata



Cellulomonas species



Chlamydia species



Chromobacterium violaceum



Chryseobacterium (Flavobacterium)



indologenes



Chryseobacterium (Flavobacterium)



meningosepticum



Chryseobacterium gleum



Chryseobacterium species



Chryseomonas indologenes



Citeromyces matritensis



Citrobacter amalonaticus



Citrobacter braakii



Citrobacter diversus



Citrobacter farmeri



Citrobacter freundii



Citrobacter freundii complex



Citrobacter koseri



Citrobacter sedlakii



Citrobacter species



Citrobacter werkmanii



Citrobacter youngae



Clostridium acetobutylicum



Clostridium barati



Clostridium beijerinckii



Clostridium bifermentans



Clostridium botulinum



Clostridium botulinum (NP) B&F



Clostridium botulinum (NP) E



Clostridium botulinum (P) A&H



Clostridium botulinum (P) F



Clostridium botulinum G1



Clostridium botulinum G2



Clostridium butyricum



Clostridium cadaveris



Clostridium chauvoei



Clostridium clostridiiforme



Clostridium difficile



Clostridium fallax



Clostridium glycolicum



Clostridium hastiforme



Clostridium histolyticum



Clostridium innocuum



Clostridium limosum



Clostridium novyi



Clostridium novyi A



Clostridium paraputrificum



Clostridium perfringens



Clostridium putrificum



Clostridium ramosum



Clostridium septicum



Clostridium sordellii



Clostridium species



Clostridium sphenoides



Clostridium sporogenes



Clostridium subterminale



Clostridium tertium



Clostridium tetani



Clostridium tyrobutyricum



Comamonas (Pseudomonas)



acidovorans



Comamonas (Pseudomonas)



testosteroni



Comamonas species



Corynebacterium accolens



Corynebacterium afermentans



Corynebacterium amycolatum



Corynebacterium aquaticum



Corynebacterium argentoratense



Corynebacterium auris



Corynebacterium bovis



Corynebacterium coyleae



Corynebacterium cystitidis



Corynebacterium diphtheriae



Corynebacterium diphtheriae biotype



belfanti



Corynebacterium diphtheriae biotype



gravis



Corynebacterium diphtheriae biotype



intermedius



Corynebacterium diphtheriae biotype



mitis



Corynebacterium flavescens



Corynebacterium glucuronolyticum



Corynebacterium glucuronolyticum-



seminale



Corynebacterium group A



Corynebacterium group A-4



Corynebacterium group A-5



Corynebacterium group ANF



Corynebacterium group B



Corynebacterium group B-3



Corynebacterium group F



Corynebacterium group F-1



Corynebacterium group F-2



Corynebacterium group G



Corynebacterium group G-1



Corynebacterium group G-2



Corynebacterium group I



Corynebacterium group I-2



Corynebacterium jeikeium (group JK)



Corynebacterium kutscheri (C. murium)



Corynebacterium macginleyi



Corynebacterium minutissimum



Corynebacterium pilosum



Corynebacterium propinquum



Corynebacterium



pseudodiphtheriticum



Corynebacterium pseudotuberculosis



Corynebacterium pyogenes



Corynebacterium renale



Corynebacterium renale group



Corynebacterium seminale



Corynebacterium species



Corynebacterium striatum (C. flavidum)



Corynebacterium ulcerans



Corynebacterium urealyticum (group



D2)



Corynebacterium xerosis



Cryptococcus albidus



Cryptococcus ater



Cryptococcus cereanus



Cryptococcus gastricus



Cryptococcus humicolus



Cryptococcus lactativorus



Cryptococcus laurentii



Cryptococcus luteolus



Cryptococcus melibiosum



Cryptococcus neoformans



Cryptococcus species



Cryptococcus terreus



Cryptococcus uniguttulatus



Debaryomyces hansenii



Debaryomyces marama



Debaryomyces polymorphus



Debaryomyces species



Dermabacter hominis



Dermacoccus (Micrococcus)



nishinomiyaensis



Dietzia species



Edwardsiella hoshinae



Edwardsiella ictaluri



Edwardsiella species



Edwardsiella tarda



Eikenella corrodens



Empedobacter brevis (Flavobacterium



breve)



Enterobacter aerogenes



Enterobacter agglomerans



Enterobacter amnigenus



Enterobacter amnigenus asburiae



(CDC enteric group 17)



Enterobacter amnigenus biogroup 1



Enterobacter amnigenus biogroup 2



Enterobacter asburiae



Enterobacter cancerogenus



Enterobacter cloacae



Enterobacter gergoviae



Enterobacter hormaechei



Enterobacter intermedius



Enterobacter sakazakii



Enterobacter species



Enterobacter taylorae



Enterobacter taylorae (CDC enteric



group 19)



Enterococcus (Streptococcus)



cecorum



Enterococcus (Streptococcus)



faecalis (Group D)



Enterococcus (Streptococcus)



faecium (Group D)



Enterococcus (Streptococcus)



saccharolyticus



Enterococcus avium (Group D)



Enterococcus casseliflavus



(Steptococcus faecium subsp.



casseliflavus)



Enterococcus durans (Streptococcus



faecium subsp. durans) (Group D)



Enterococcus gallinarum



Enterococcus hirae



Enterococcus malodoratus



Enterococcus mundtii



Enterococcus raffinosus



Enterococcus species



Erwinia amylovora



Erwinia carotovora



Erwinia carotovora subsp. atroseptica



Erwinia carotovora subsp.



betavasculorum



Erwinia carotovora subsp. carotovora



Erwinia chrysanthemi



Erwinia cypripedii



Erwinia mallotivora



Erwinia nigrifluens



Erwinia quercina



Erwinia rhapontici



Erwinia rubrifaciens



Erwinia salicis



Erwinia species



Erysipelothrix rhusiopathiae



Erysipelothrix species



Escherichia blattae



Escherichia coli



Escherichia coli A-D



Escherichia coli O157:H7



Escherichia fergusonii



Escherichia hermannii



Escherichia species



Escherichia vulneris



Eubacterium aerofaciens



Eubacterium alactolyticum



Eubacterium lentum



Eubacterium limosum



Eubacterium species



Ewingella americana



Filobasidiella neoformans



Filobasidium floriforme



Filobasidium uniguttulatum



Flavimonas oryzihabitans



Flavobacterium gleum



Flavobacterium indologenes



Flavobacterium odoratum



Flavobacterium species



Francisella novicida



Francisella philomiragia



Francisella species



Francisella tularensis



Fusobacterium mortiferum



Fusobacterium necrogenes



Fusobacterium necrophorum



Fusobacterium nucleatum



Fusobacterium species



Fusobacterium varium



Gaffkya species



Gardnerella vaginalis



Gemella haemolysans



Gemella morbillorum



Gemella species



Geotrichum candidum



Geotrichum fermentans



Geotrichum penicillarum



Geotrichum penicillatum



Geotrichum species



Gordona species



Haemophilus aegyptius



Haemophilus aphrophilus



Haemophilus ducreyi



Haemophilus haemoglobinophilus



Haemophilus haemolyticus



Haemophilus influenzae



Haemophilus influenzae biotype I



Haemophilus influenzae biotype II



Haemophilus influenzae biotype III



Haemophilus influenzae biotype IV



Haemophilus influenzae biotype V



Haemophilus influenzae biotype VI



Haemophilus influenzae biotype VII



Haemophilus influenzae biotype VIII



Haemophilus paragallinarum



Haemophilus parahaemolyticus



Haemophilus parainfluenzae



Haemophilus parainfluenzae biotype I



Haemophilus parainfluenzae biotype II



Haemophilus parainfluenzae biotype



III



Haemophilus parainfluenzae biotype



IV



Haemophilus parainfluenzae biotype V



Haemophilus parainfluenzae biotype



VI



Haemophilus parainfluenzae biotype



VII



Haemophilus parainfluenzae biotype



VIII



Haemophilus paraphrohaemolyticus



Haemophilus paraphrophilus



Haemophilus segnis



Haemophilus somnus



Haemophilus species



Hafnia alvei



Hanseniaspora guilliermondii



Hanseniaspora uvarum



Hanseniaspora valbyensis



Hansenula anomala



Hansenula holstii



Hansenula polymorpha



Helicobacter (Campylobacter) cinaedi



Helicobacter (Campylobacter)



fennelliae



Helicobacter (Campylobacter) pylori



Issatchenkia orientalis



Kingella denitrificans



Kingella indologenes



Kingella kingae



Kingella species



Klebsiella ornithinolytica



Klebsiella oxytoca



Klebsiella planticola



Klebsiella pneumoniae subsp.



ozaenae



Klebsiella pneumoniae subsp.



pneumoniae



Klebsiella pneumoniae subsp.



rhinoscleromatis



Klebsiella species



Klebsiella terrigena



Kloeckera apiculata



Kloeckera apis



Kloeckera japonica



Kloeckera species



Kluyvera ascorbata



Kluyvera cryocrescens



Kluyvera species



Kluyveromyces lactis



Kluyveromyces marxianus



Kluyveromyces thermotolerans



Kocuria (Micrococcus) kristinae



Kocuria (Micrococcus) rosea



Kocuria (Micrococcus) varians



Koserella trabulsii



Kytococcus (Micrococcus)



sedentarius



Lactobacillus (Weissella) viridescens



Lactobacillus A



Lactobacillus acidophilus



Lactobacillus B



Lactobacillus brevis



Lactobacillus buchneri



Lactobacillus casei



Lactobacillus casei subsp. casei



Lactobacillus casei subsp. lactosus



Lactobacillus casei subsp. rhamnosus



Lactobacillus catenaformis



Lactobacillus cellobiosus



Lactobacillus collinoides



Lactobacillus coprophilus



Lactobacillus crispatus



Lactobacillus curvatus



Lactobacillus delbrueckii subsp.



bulgaricus



Lactobacillus delbrueckii subsp.



delbrueckii



Lactobacillus delbrueckii subsp. lactis



Lactobacillus fermentum



Lactobacillus fructivorans



Lactobacillus helveticus



Lactobacillus helveticus subsp. jugurti



Lactobacillus jensenii



Lactobacillus lindneri



Lactobacillus minutus



Lactobacillus paracasei subsp.



paracasei



Lactobacillus pentosus



Lactobacillus plantarum



Lactobacillus salivarius



Lactobacillus salivarius var. salicinius



Lactobacillus species



Lactococcus diacitilactis



Lactococcus garvieae



Lactococcus lactis subsp. cremoris



Lactococcus lactis subsp. diacitilactis



Lactococcus lactis subsp. hordniae



Lactococcus lactis subsp. lactis



Lactococcus plantarum



Lactococcus raffinolactis



Leclercia adecarboxylata



Legionella species



Leminorella species



Leptospira species



Leptotrichia buccalis



Leuconostoc (Weissella)



paramesenteroides



Leuconostoc carnosum



Leuconostoc citreum



Leuconostoc gelidum



Leuconostoc lactis



Leuconostoc mesenteroides



Leuconostoc mesenteroides subsp.



cremoris



Leuconostoc mesenteroides subsp.



dextranicum



Leuconostoc mesenteroides subsp.



mesenteroides



Leuconostoc species



Listeria grayi



Listeria innocua



Listeria ivanovii



Listeria monocytogenes



Listeria murrayi



Listeria seeligeri



Listeria species



Listeria welshimeri



Megasphaera elsdenii



Methylobacterium mesophilicum



Metschnikowia pulcherrima



Microbacterium species



Micrococcus luteus



Micrococcus lylae



Micrococcus species



Mobiluncus curtisii



Mobiluncus mulieris



Mobiluncus species



Moellerella wisconsensis



Moraxella (Branhamella) catarrhalis



Moraxella atlantae



Moraxella bovis



Moraxella lacunata



Moraxella nonliquefaciens



Moraxella osloensis



Moraxella phenylpyruvica



Moraxella species



Morganella morganii



Morganella morganii subsp. morganii



Morganella morganii subsp. sibonii



Mycobacterium africanum



Mycobacterium asiaticum



Mycobacterium avium



Mycobacterium bovis



Mycobacterium chelonae



Mycobacterium fortuitum



Mycobacterium gordonae



Mycobacterium kansasii



Mycobacterium malmoense



Mycobacterium marinum



Mycobacterium phlei



Mycobacterium scrofulaceum



Mycobacterium smegmatis



Mycobacterium species



Mycobacterium tuberculosis



Mycobacterium ulcerans



Mycobacterium xenopi



Mycoplasma fermentans



Mycoplasma hominis



Mycoplasma orale



Mycoplasma pneumoniae



Mycoplasma species



Myroides species



Neisseria cinerea



Neisseria elongata subsp. elongata



Neisseria flava



Neisseria flavescens



Neisseria gonorrhoeae



Neisseria lactamica



Neisseria meningitidis



Neisseria mucosa



Neisseria perflava



Neisseria polysaccharea



Neisseria saprophytes



Neisseria sicca



Neisseria subflava



Neisseria weaveri



Neisseria weaveri (CDC group M5)



Nocardia species



Ochrobactrum anthropi



Oerskovia species



Oerskovia xanthineolytica



Oligella (Moraxella) urethralis



Oligella species



Oligella ureolytica



Paenibacillus alvei



Paenibacillus macerans



Paenibacillus polymyxa



Pantoea agglomerans



Pantoea ananas (Erwinia uredovora)



Pantoea dispersa



Pantoea species



Pantoea stewartii



Pasteurella (Haemophilus) avium



Pasteurella aerogenes



Pasteurella gallinarum



Pasteurella haemolytica



Pasteurella haemolyticus



Pasteurella multocida



Pasteurella multocida SF



Pasteurella multocida subsp.



multocida



Pasteurella multocida subsp. septica



Pasteurella pneumotropica



Pasteurella species



Pasteurella ureae



Pediococcus acidilactici



Pediococcus damnosus



Pediococcus pentosaceus



Pediococcus species



Peptococcus niger



Peptococcus species



Peptostreptococcus anaerobius



Peptostreptococcus asaccharolyticus



Peptostreptococcus indolicus



Peptostreptococcus magnus



Peptostreptococcus micros



Peptostreptococcus parvulus



Peptostreptococcus prevotii



Peptostreptococcus productus



Peptostreptococcus species



Peptostreptococcus tetradius



Phaecoccomyces exophialiae



Photobacterium damselae



Pichia (Hansenula) anomala



Pichia (Hansenula) jadinii



Pichia (Hansenula) petersonii



Pichia angusta (Hansenula



polymorpha)



Pichia carsonii (P. vini)



Pichia etchellsii



Pichia farinosa



Pichia fermentans



Pichia membranaefaciens



Pichia norvegensis



Pichia ohmeri



Pichia spartinae



Pichia species



Plesiomonas shigelloides



Porphyromonas asaccharolytica



Porphyromonas endodontalis



Porphyromonas gingivalis



Porphyromonas levii



Provotella (Bacteroides) buccae



Prevotella (Bacteroides) buccalis



Prevotella (Bacteroides) corporis



Pravotella (Bacteroides) denticola



Prevotella (Bacteroides) loescheii



Prevotella (Bacteroides) oralis



Prevotella (Bacteroides) disiens



Prevotella (Bacteroides) oris



Prevotella bivia (Bacteroides bivius)



Prevotella intermedia (Bacteroides



intermedius)



Prevotella melaninogenica



(Bacteroides melaninogenicus)



Prevotella ruminicola



Propionibacterium acnes



Propionibacterium avidum



Propionibacterium granulosum



Propionibacterium propionicum



Propionibacterium species



Proteus mirabilis



Proteus penneri



Proteus species



Proteus vulgaris



Prototheca species



Prototheca wickerhamii



Prototheca zopfii



Providencia alcalifaciens



Providencia heimbachae



Providencia rettgeri



Providencia rustigianii



Providencia species



Providencia stuartii



Providencia stuartii urea +



Pseudomonas (Chryseomonas)



luteola



Pseudomonas acidovorans



Pseudomonas aeruginosa



Pseudomonas alcaligenes



Pseudomonas cepacia



Pseudomonas chlororaphis (P. aureofaciens)



Pseudomonas fluorescens



Pseudomonas fluorescens group



Pseudomonas mendocina



Pseudomonas pseudoalcaligenes



Pseudomonas putida



Pseudomonas species



Pseudomonas stutzeri



Pseudomonas testosteroni



Pseudomonas vesicularis



Pseudoramibacter (Eubacterium)



alactolyticus



Psychrobacter (Moraxella)



phenylpyruvicus



Rahnella aquatilis



Ralstonia (Pseudomonas,



Burkholderia) pickettii



Rhodococcus (Corynebacterium) equi



Rhodococcus species



Rhodosporidium toruloides



Rhodotorula glutinis



Rhodotorula minuta



Rhodotorula mucilaginosa (R. rubra)



Rhodotorula species



Rickettsia species



Rothia dentocariosa



Saccharomyces cerevisiae



Saccharomyces exiguus



Saccharomyces kluyverii



Saccharomyces species



Sakaguchia dacryoides



(Rhodosporidium dacryoidum)



Salmonella arizonae



Salmonella choleraesuis



Salmonella enteritidis



Salmonella gallinarum



Salmonella paratyphi A



Salmonella paratyphi B



Salmonella pullorum



Salmonella species



Salmonella typhi



Salmonella typhimurium



Salmonella typhisuis



Salmonella/Arizona



Serratia ficaria



Serratia fonticola



Serratia grimesii



Serratia liquefaciens



Serratia marcescens



Serratia odorifera



Serratia odorifera type 1



Serratia odorifera type 2



Serratia plymuthica



Serratia proteamaculans



Serratia proteamaculans subsp.



proteamaculans



Serratia proteamaculans subsp.



quinovora



Serratia rubidaea



Serratia species



Shewanella (Pseudomonas,



Alteromonas) putrefaciens



Shigella boydii



Shigella dysenteriae



Shigella flexneri



Shigella sonneri



Shigella species



Sphingobacterium multivorum



Sphingobacterium species



Sphingobacterium spiritivorum



Sphingobacterium thalpophilum



Sphingomonas (Pseudomonas)



paucimobilis



Sporidiobolus salmonicolor



Sporobolomyces roseus



Sporobolomyces salmonicolor



Sporobolomyces species



Staphylococcus (Peptococcus)



saccharolyticus



Staphylococcus arlettae



Staphylococcus aureus



Staphylococcus aureus (Coagulase-



negative)



Staphylococcus auricularis



Staphylococcus capitis



Staphylococcus capitis subsp. capitis



Staphylococcus capitis subsp.



ureolyticus



Staphylococcus caprae



Staphylococcus carnosus



Staphylococcus caseolyticus



Staphylococcus chromogenes



Staphylococcus cohnii



Staphylococcus cohnii subsp. cohnii



Staphylococcus cohnii subsp.



urealyticum



Staphylococcus epidermidis



Staphylococcus equorum



Staphylococcus gallinarum



Staphylococcus haemolyticus



Staphylococcus hominis



Staphylococcus hominis subsp.



hominis



Staphylococcus hominis subsp.



novobiosepticus



Staphylococcus hyicus



Staphylococcus intermedius



Staphylococcus kloosii



Staphylococcus lentus



Staphylococcus lugdunensis



Staphylococcus saprophyticus



Staphylococcus schleiferi



Staphylococcus sciuri



Staphylococcus simulans



Staphylococcus species



Staphylococcus warneri



Staphylococcus xylosus



Stenotrophomonas (Xanthomonas)



maltophilia



Stephanoascus ciferrii



Stomatococcus mucilaginosus



Streptococcus acidominimus



Streptococcus agalactiae



Streptococcus agalactiae (Group B)



Streptococcus agalactiae hemolytic



Streptococcus agalactiae non-



hemolytic



Streptococcus alactolyticus



Streptococcus anginosus



Streptococcus anginosus (Group D,



nonenterococci)



Streptococcus beta-hemolytic group A



Streptococcus beta-hemolytic non-



group A or B



Streptococcus beta-hemolytic non-



group A



Streptococcus beta-hemolytic



Streptococcus bovis (Group D,



nonenterococci)



Streptococcus bovis I



Streptococcus bovis II



Streptococcus canis



Streptococcus constellatus



Streptococcus constellatus



(Streptococcus milleri I)



Streptococcus constellatus (viridans



Streptococcus)



Streptococcus downei



Streptococcus dysgalactiae subsp.



dysgalactiae



Streptococcus dysgalactiae subsp.



equisimilis



Streptococcus equi (Group C/Group G



Streptococcus)



Streptococcus equi subsp. equi



Streptococcus equi subsp.



zooepidemicus



Streptococcus equinus



Streptococcus equinus (Group D,



nonenterococci)



Streptococcus equisimilis



Streptococcus equisimulis (Group



C/Group G Streptococcus)



Streptococcus Gamma (non)-



hemolytic



Streptococcus gordonii



Streptococcus Group B



Streptococcus Group C



Streptococcus Group D



Streptococcus Group E



Streptococcus Group F



Streptococcus Group G



Streptococcus Group L



Streptococcus Group P



Streptococcus Group U



Streptococcus intermedius



Streptococcus intermedius



(Streptococcus milleri II)



Streptococcus intermedius (viridans



Streptococcus)



Streptococcus milleri group



Streptococcus mitis



Streptococcus mitis (viridans



Streptococcus)



Streptococcus mitis group



Streptococcus mutans



Streptococcus mutans (viridans



Streptococcus)



Streptococcus oralis



Streptococcus parasanguis



Streptococcus pneumoniae



Streptococcus porcinus



Streptococcus pyogenes



Streptococcus pyogenes (Group A)



Streptococcus salivarius



Streptococcus salivarius (viridans



Streptococcus)



Streptococcus salivarius subsp.



salivarius



Streptococcus salivarius subsp.



thermophilus



Streptococcus sanguis



Streptococcus sanguis I (viridans



Streptococcus)



Streptococcus sanguis II



Streptococcus sanguis II (viridans



Streptococcus)



Streptococcus sobrinus



Streptococcus species



Streptococcus suis I



Streptococcus suis II



Streptococcus uberis



Streptococcus uberis (viridans



Streptococcus)



Streptococcus vestibularis



Streptococcus zooepidemicus



Streptococcus zooepidemicus (Group



C)



Streptomyces somaliensis



Streptomyces species



Suttonella (Kingella) indologenes



Tatumella ptyseos



Tetragenococcus (Pediococcus)



halophilus



Torulaspora delbrueckii



(Saccharomyces rosei)



Torulopsis candida



Torulopsis haemulonii



Torulopsis inconspicua



Treponema species



Trichosporon asahii



Trichosporon asteroides



Trichosporon beigelii



Trichosporon cutaneum



Trichosporon inkin



Trichosporon mucoides



Trichosporon ovoides



Trichosporon pullulans



Trichosporon species



Turicella otitidis



Ureaplasma species



Ureaplasma urealyticum



Veillonella parvula (V. alcalescens)



Veillonella species



Vibrio alginolyticus



Vibrio cholerae



Vibrio damsela



Vibrio fluvialis



Vibrio furnissii



Vibrio harveyi



Vibrio hollisae



Vibrio metschnikovii



Vibrio mimicus



Vibrio parahaemolyticus



Vibrio species



Vibrio species SF



Vibrio vulnificus



Weeksella (Bergeylla) virosa



Weeksella species



Weeksella virosa



Williopsis (Hansenula) satumus



Xanthomonas campestris



Xanthomonas species



Yarrowia (Candida) lipolytica



Yersinia aldovae



Yersinia enterocolitica



Yersinia enterocolitica group



Yersinia frederiksenii



Yersinia intermedia



Yersinia intermedius



Yersinia kristensenii



Yersinia pestis



Yersinia pseudotuberculosis



Yersinia pseudotuberculosis SF



Yersinia ruckeri



Yersinia species



Yokenella regensburgei



Yokenella regensburgei (Koserella



trabulsii)



Zygoascus hellenicus



Zygosaccharomyces species







1The list includes microorganisms that may be identified by API identification test systems and VITEK ® automated identification system from bioMérieux Inc., or by the MicroScan ®-WalkAway ® automated systems from Dade Behring. Identification relies on classical identification methods using batteries of biochemical and other phenotypical tests.













TABLE 16







tuf gene sequences obtained in our laboratory (Example 42).













GenBank


Species
Strain no.
Gene
Accession no.*





Abiotrophia adiacens
ATCC49175
tuf
AF124224


Enterococcus avium
ATCC14025
tufA
AF124220




tufB
AF274715


Enterococcus casseliflavus
ATCC25788
tufA
AF274716




tufB
AF274717


Enterococcus cecorum
ATCC43198
tuf
AF274718


Enterococcus columbae
ATCC51263
tuf
AF274719


Enterococcus dispar
ATCC51266
tufA
AF274720




tufB
AF274721


Enterococcus durans
ATCC19432
tufA
AF274722




tufB
AF274723


Enterococcus faecalis
ATCC29212
tuf
AF124221


Enterococcus faecium
ATCC 19434
tufA
AF124222




tufB
AF274724


Enterococcus gallinarum
ATCC49573
tufA
AF124223




tufB
AF274725


Enterococcus hirae
ATCC8043
tufA
AF274726




tufB
AF274727


Enterococcus malodoratus
ATCC43197
tufA
AF274728




tufB
AF274729


Enterococcus mundtii
ATCC43186
tufA
AF274730




tufB
AF274731


Enterococcus pseudoavium
ATCC49372
tufA
AF274732




tufB
AF274733


Enterococcus raffinosus
ATCC49427
tufA
AF274734




tufB
AF274735


Enterococcus saccharolyticus
ATCC43076
tuf
AF274736


Enterococcus solitarius
ATCC49428
tuf
AF274737


Enterococcus sulfureus
ATCC49903
tuf
AF274738


Lactococcus lactis
ATCC11154
tuf
AF274745


Listeria monocytogenes
ATCC15313
tuf
AF274746


Listeria seeligeri
ATCC35967
tuf
AF274747


Staphylococcus aureus
ATCC25923
tuf
AF274739


Staphylococcus epidermidis
ATCC14990
tuf
AF274740


Streptococcus mutans
ATCC25175
tuf
AF274741


Streptococcus pneumoniae
ATCC6303
tuf
AF274742


Streptococcus pyogenes
ATCC19615
tuf
AF274743


Streptococcus suis
ATCC43765
tuf
AF274744





*Corresponding sequence ID NO. for the above ATCC strains are given in table 7.













TABLE 17







tuf gene sequences selected from databases for Example 42.











Species
Gene
Accession no.*







Agrobacterium tumefaciens
tufA
X99673




tufB
X99674



Anacystis nidulans
tuf
X17442



Aquifex aeolicus
tufA
AE000657




tufB
AE000657



Bacillus stearothermophilus
tuf
AJ000260



Bacillus subtilis
tuf
AL009126



Bacteroides fragilis
tuf
P33165



Borrelia burgdorferi
tuf
AE000783



Brevibacterium linens
tuf
X76863



Bulkholderia cepacia
tuf
P33167



Campylobacter jejuni
tufB
Y17167



Chlamydia pneumoniae
tuf
AE001363



Chlamydia trachomatis
tuf
M74221



Corynebacterium glutamicum
tuf
X77034



Cytophaga lytica
tuf
X77035



Deinococcus radiodurans
tuf
AE000513



Escherichia coli
tufA
J01690




tufB
J01717



Fervidobacterium islandicum
tuf
Y15788



Haemophilus influenzae
tufA
L42023




tufB
L42023



Helicobacter pylori
tuf
AE000511



Homo sapiens (Human)
EF-1α
X03558



Methanococcus jannaschii
EF-1α
U67486



Mycobacterium leprae
tuf
D13869



Mycobacterium tuberculosis
tuf
X63539



Mycoplasma genitalium
tuf
L43967



Mycoplasma pneumoniae
tuf
U00089



Neisseria gonorrhoeae
tufA
L36380



Nicotiana tabacum (Tobacco)
EF-1α
U04632



Peptococcus niger
tuf
X76869



Planobispora rosea
tuf1
U67308



Saccharomyces cerevisiae (Yeast)
EF-1α
X00779



Salmonella typhimurium
tufA
X55116




tufB
X55117



Shewanella putrefaciens
tuf
P33169



Spirochaeta aurantia
tuf
X76874



Spirulina platensis
tufA
X15646



Streptomyces aureofaciens
tuf1
AF007125



Streptomyces cinnamoneus
tuf1
X98831



Streptomyces coelicolor
tuf1
X77039




tuf3
X77040



Streptomyces collinus
tuf1
S79408



Streptomyces ramocissimus
tuf1
X67057




tuf2
X67058




tuf3
X67059



Synechocystis sp.
tuf
AB001339



Taxeobacter ocellatus
tuf
X77036



Thermotoga maritima
tuf
AE000512



Thermus aquaticus
tuf
X66322



Thermus thermophilus
tuf
X06657



Thiobacillus cuprinus
tuf
U78300



Treponema pallidum
tuf
AE000520



Wolinella succinogenes
tuf
X76872







*Sequence data were obtained from GenBank, EMBL, and SWISSPROT databases. Genes were designated as appeared in the references.













TABLE 18





Nucleotide and amino acid sequence identities of EF-Tu between different enterococci and other low G + C gram-positive bacteria.
















The upper right triangle represents the deduced amino acid sequence identities of gram-positive bacterial EF-Tu, while the lower left triangle represents the DNA sequence identities of the corresponding tuf genes. The sequence identities between different enterococcal tufA genes are boxed while those between enterococcal tufB genes are shaded.













TABLE 19







Strains analyzed in Example 43.













16S rDNA





sequence





accession


Taxon
Strain*
Strain†
number





Cedecea davisae
ATCC 33431T




Cedecea lapagei
ATCC 33432T


Cedecea neteri
ATCC 33855T


Citrobacter amalonaticus
ATCC 25405T
CDC 9020-77T
AF025370


Citrobacter braakii
ATCC 43162
CDC 080-58T
AF025368


Citrobacter farmeri
ATCC 51112T
CDC 2991-81T
AF025371


Citrobacter freundii
ATCC 8090T
DSM 30039T
AJ233408


Citrobacter koseri
ATCC 27156T


Citrobacter sedlakii
ATCC 51115T
CDC 4696-86T
AF025364


Citrobacter werkmanii
ATCC 51114T
CDC 0876-58T
AF025373


Citrobacter youngae
ATCC 29935T


Edwardsiella hoshinae
ATCC 33379T


Edwardsiella tarda
ATCC 15947T
CDC 4411-68
AF015259


Enterobacter aerogenes
ATCC 13048T
JCM 1235T
AB004750


Enterobacter agglomerans
ATCC 27989


Enterobacter amnigenus
ATCC 33072T
JCM 1237T
AB004749


Enterobacter asburiae
ATCC 35953T
JCM 6051T
AB004744


Enterobacter cancerogenus
ATCC 35317T


Enterobacter cloacae
ATCC 13047T


Enterobacter gergoviae
ATCC 33028T
JCM 1234T
AB004748


Enterobacter hormaechei
ATCC 49162T


Enterobacter sakazakii
ATCC 29544T
JCM 1233T
AB004746


Escherichia coli
ATCC 11775T
ATCC 11775T
X80725


Escherichia coli
ATCC 25922
ATCC 25922
X80724


Escherichia coli (ETEC)
ATCC 35401


Escherichia coli (O157:H7)
ATCC 43895
ATCC 43895
Z83205


Escherichia fergusonii
ATCC 35469T


Escherichia hermanii
ATCC 33650T


Escherichia vulneris
ATCC 33821T
ATCC 33821T
X80734


Ewingella americana
ATCC 33852T
NCPPB 3905
X88848


Hafnia alvei
ATCC 13337T
ATCC 13337T
M59155


Klebsiella ornithinolytica
ATCC 31898
CIP 103.364
U78182


Klebsiella oxytoca
ATCC 33496
ATCC 13182T
U78183


Klebsiella planticola
ATCC 33531T
JCM 7251T
AB004755


Klebsiella pneumoniae


subsp. pneumoniae
ATCC 13883T
DSM 30104T
AJ233420


subsp. ozaenae
ATCC 11296T
ATCC 11296T
Y17654


subsp. rhinoscleromatis
ATCC 13884T


Kluyvera ascorbata
ATCC 33433T
ATCC 14236
Y07650


Kluyvera cryocrescens
ATCC 33435T


Kluyvera georgiana
ATCC 51603T


Leclercia adecarboxylata
ATCC 23216T


Leminorella grimontii
ATCC 33999T
DSM 5078T
AJ233421


Moellerella wisconsensis
ATCC 35017T


Morganella morganii
ATCC 25830T


Pantoea agglomerans
ATCC 27155T
DSM 3493T
AJ233423


Pantoea dispersa
ATCC 14589T


Plesiomonas shigelloides
ATCC 14029T


Pragia fontium
ATCC 49100T
DSM 5563T
AJ233424


Proteus mirabilis
ATCC 25933


Proteus penneri
ATCC 33519T


Proteus vulgaris
ATCC 13315T
DSM 30118T
AJ233425


Providencia alcalifaciens
ATCC 9886T


Providencia rettgeri
ATCC 9250


Providencia rustigianii
ATCC 33673T


Providencia stuartii
ATCC 33672


Rahnella aquatilis
ATCC 33071T
DSM 4594T
AJ233426


Salmonella choleraesuis


subsp. arizonae
ATCC 13314T


subsp. choleraesuis


serotype Choleraesuis
ATCC 7001


serotype Enteritidis‡
ATCC 13076T
SE22
SE22


serotype Gallinarum
ATCC 9184


serotype Heidelberg
ATCC 8326


serotype Paratyphi A
ATCC 9150


serotype Paratyphi B
ATCC 8759


serotype Typhi‡
ATCC 10749
St111
U88545


serotype Typhimurium‡
ATCC 14028


serotype Virchow
ATCC 51955


subsp. diarizonae
ATCC 43973T


subsp. houtenae
ATCC 43974T


subsp. indica
ATCC 43976T


subsp. salamae
ATCC 43972T


Serratia fonticola
DSM 4576T
DSM 4576T
AJ233429


Serratia grimesii
ATCC 14460T
DSM 30063T
AJ233430


Serratia liquefaciens
ATCC 27592T


Serratia marcescens
ATCC 13880T
DSM 30121T
AJ233431


Serratia odorifera
ATCC 33077T
DSM 4582T
AJ233432


Serratia plymuthica
DSM 4540T
DSM 4540T
AJ233433


Serratia rubidaea
DSM 4480T
DSM 4480T
AJ233436


Shigella boydii
ATCC 9207
ATCC 9207
X96965


Shigella dysenteriae
ATCC 11835
ATCC 13313T
X96966




ATCC 25931
X96964


Shigella flexneri
ATCC 12022
ATCC 12022
X96963


Shigella sonnei
ATCC 29930T


Tatumella ptyseos
ATCC 33301T
DSM 5000T
AJ233437


Trabulsiella guamensis
ATCC 49490T


Yersinia enterocolitica
ATCC 9610T
ATCC 9610T
M59292


Yersinia frederiksenii
ATCC 33641T


Yersinia intermedia
ATCC 29909T


Yersinia pestis
RRB KIMD27
ATCC 19428T
X75274


Yersinia pseudotuberculosis
ATCC 29833T


Yersinia rohdei
ATCC 43380T
ER-2935T
X75276


Shewanella putrefaciens
ATCC 8071T


Vibrio cholerae
ATCC 25870
ATCC 14035T
X74695





T Type strain


*Strains used in this study for sequencing of partial tuf and atpD genes. SEQ ID NOs. for tuf and atpD sequences corresponding to the above reference strains are given in table 7.


†Strains used in other studies for sequencing of 16S rDNA gene. When both strain numbers are on the same row, both strains are considered to be the same although strain numbers may be different.


‡Phylogenetic serotypes considered species by the Bacteriological Code (1990 Revision).













TABLE 20







PCR primer pairs used in this study










Primer

Nucleotide
Amplicon


SEQ ID NO.
Sequence
positions*
length (bp)





tuf





664
5′-AAYATGATIACIGGIGCIGCICARATGGA-3′
271-299
884


697
5′-CCIACIGTICKICCRCCYTCRCG-3′
1132-1156



 


atpD





568
5′-RTIATIGGIGCIGTIRTIGAYGT-3′
25-47
884


567
5′-TCRTCIGCIGGIACRTAIAYIGCYTG-3′
883-908



 


700
5′-TIRTIGAYGTCGARTTCCCTCARG-3′
38-61
871


567
5′-TCRTCIGCIGGIACRTAIAYIGCYTG-3′
883-908





*The nudeotide positions given are for E. coli tuf and atpD sequences (GenBank accession no. AE000410 and V00267, respectively). Numbering starts from the first base of the initiation codon.













TABLE 21







Selection of M. catarrhalis-specific primer pairs from SEQ ID NO:291


(466 bp DNA fragment) other than those previously tested2.

























Mor-
Mor-
Mor-















ax-
ax-
ax



Mor-











ella
ella
ella



ax-
King-

Neiss-







Amp-
cat-
cat-
non-
Mor-
Mor-
Mor-
ella
ella 
eria
Neiss-

ylo-
Staph-




li-
arr-
arr-
li-
ax-
ax-
ax-
phen-
in-
King-
men-
eria
Esc-
coc-




con
halis
halis
que-
ella
ella
ella
yl-
do-
ella
in-
gon-
her-
cus




size
ATCC
ATCC
fac-
lacu-
oslo-
atla-
pyru-
lo-
king-
giti-
orr-
ichia
aur-


Primer
Sequence
(bp)
43628
53879
iens
nata
ensis
ntae
vica
genes
ea
dis
hoeae
coli
eus





SEQ ID
CGCTGACG
118
+3
+
−
−
−
−
−
−
−
−
−
−
−


NO: 118
GCTTGTTT

















GTACCA
















SEQ ID
TGTTTTGA
















NO: 119
GCTTTTTA

















TTTTTTGA
















 


VB  
TGCTTAAG
93
+
+
−
−
−
−
−
−
−
−
−
−
−


mcat1
ATTCACTC
















(SEQ 
TGCCATTT
















ID NO:
T
















2298)

















VB
TAAGTCGC
















mcat2
TGACGGCT
















(SEQ 
TGTTT
















ID NO: 

















2299)

















 


VB
CCTGCACC
140
+
+
−
−
−
−
−
−
−
−
−
−
−


mcat3
ACAAGTCA
















(SEQ 
TCAT
















ID NO: 

















2300)

















VB
AATTCACC
















mcat4
AACAATGT
















(SEQ
CAAAGC
















ID NO: 

















2301)

















 


VB
AATGATAA
219
+
+
−
−
−
−
−
−
−
−
−
−
−


mcat5
CCAGTCAA
















(SEQ 
GCAAGC
















ID NO:

















2302)

















VB
GGTGCATG
















mcat6
GTGATTTG
















(SEQ 
TAAAA
















ID NO:

















2303)

















 


VB
GTGTGCGT
160
+
+
−
−
−
−
−
−
−
−
−
−
− 


mcat7
TCACTTTT
















(SEQ 
ACAAAT
















ID NO:

















2304)

















VB
GGTGTTAA
















mcat8
GCTGATGA
















(SEQ 
TGAGAG
















ID NO:

















2305)

















 


VB
TGACCATG
167
+
+
−
−
−
−
−
−
−
−
−
−
−


mcat9
CACACCCT
















(SEQ 
TATT
















ID NO:

















2306)

















VB
TCATTGGG
















mcat10
ATGAAAGT
















(SEQ 
ATCGTT
















ID NO:

















2307)





1SEQ ID NO. from U.S. pat. 6,001,564.


2All PCR assays were performed with 1 ng of purified genomic DNA by using an annealing temperature of 55° C. and 30 cycles of amplification. The genomic DNA from the various bacterial species above was always isolated from reference strains obtained from ATCC.


3All positive results showed a strong amplification signal with genomic DNA from the target species M. catarrhalis.













TABLE 22





Selection of S. epidermidis-speciflc primer pairs from SEQ ID NO:361


(705 bp DNA fragment) other than those previously tested.



























Staphyl-
Staphyl-










ococcus
ococcus









Ampli-
epiderm-
epiderm-



Staphyl-





con
idis,
idis,
Staphyl-
Staphyl-
Staphyl-
ococcus




Sequence
size
ATCC
ATCC
ococcus
ococcus
ococcus
auri-
Staphyl-


Primer
(all 25 nucleotides)
(bp)
14990
12228
capitis
cohnii
aureus
cularis
ococcus





SEQ ID 
ATCAAAAAGTTGGCGAACCTTTTCA
125
+3
+
−
−
−
−
−


NO: 145











SEQ ID 
CAAAAGAGCGTGGAGAAAAGTATCA










NO: 146











 


VBsep3  
CATAGTCTGATTGCTCAAAGTCTTG
208
+
+
−
−
−
−
+


(SEQ











ID NO:











2308)











VBsep4
GCGAATAGTGAACTACATTCTGTTG

+
+
−
−
−
−
−


(SEQ 











ID NO:











2309)











 


VBsep5
CACGCTCTTTTGCAATTTCCATTGA
208
+
+
+
+
+
−
+


(SEQ











ID NO:











2310)











VBsep6
GAAGCAAATATTCAAAATGCACCAG

+
+
+
+
+
−
+


(SEQ











ID NO:











2311)











 


VBsep7
AAAGTCTTTTGCTTCTTCAGATTCA
177
+
+
−
−
−
−
+


(SEQ











ID NO:











2312)











VBsep8
GTGTTCACAGGTATGGATGCTCTTA

+
+
NT
NT
−
NT
−


(SEQ


+
+
NT
NT
−
NT
−


ID NO:











2313)











 


VBsep9
GAGCATCCATACCTGTGAACACAGA
153
+
+
−
−
−
−
+


(SEQ











ID NO:











2314)











VBsep10
TTTTCCAATTACAAGAGACATCAGT

+
+
NT
NT
−
NT
+


(SEQ


+
+
NT
NT
−
NT
−


ID NO:











2315)











 


VBsep11
TTTGAATTCGCATGTACTTTGTTTG
135
+
+
−
−
−
−
−


(SEQ 











ID NO:











2316)











VBsep12
CCCCGGGTTCGAAATCGATAAAAAG










(SEQ











ID NO:











2317)







Ampli-











con
Staphyl-


Staphyl-
Staphyl-
Bacillus
Entero-



Sequence
size
ococcus
Staphyl-
Stahyl-
ococcus
occoccus
stub-
coccus


Primer
(all 25 nucleotides)
(bp)
hominis
ococcus
ococcus
simulans
warneri
tilis
faecalis





SEQ ID
ATCAAAAAGTTGGCGAACCTTTTCA
125
−
−
−
−
−
−
−


NO: 145











SEQ ID
CAAAAGAGCGTGGAGAAAAGTATCA










NO: 146











 


VBsep3
CATAGTCTGATTGCTCAAAGTCTTG
208
−
−
−
−
−
−
−


(SEQ











ID NO:











2308)











VBsep4
GCGAATAGTGAACTACATTCTGTTG

−
−
−
−
−
−
−


(SEQ











ID NO:











2309)











 


VBsep5
CACGCTCTTTTGCAATTTCCATTGA
208
+
−
−
−
−
−
−


(SEQ











ID NO:











2310)











VBsep6
GAAGCAAATATTCAAAATGCACCAG

+
−
−
−
NT
NT
NT


(SEQ











ID NO:











2311)











 


VBsep7
AAAGTCTTTTGCTTCTTCAGATTCA
177
−
−
−
+
−
−
−


(SEQ











ID NO:











2312)











VBsep8
GTGTTCACAGGTATGGATGCTCTTA

NT
−
−
+
NT
NT
NT


(SEQ


NT
−
−
−
NT
NT
NT


ID NO:











2313)











 


VBsep9
GAGCATCCATACCTGTGAACACAGA
153
−
+
+
−
−
−
−


(SEQ











ID NO:











2314)











VBsep10
TTTTCCAATTACAAGAGACATCAGT

NT
+
−
−
NT
NT
NT


(SEQ


NT
−
−
−
NT
NT
NT


ID NO:











2315)











 


VBsep11
TTTGAATTCGCATGTACTTTGTTTG
135
−
−
−
−
−
−
−


(SEQ











ID NO:











2316)











VBsep12
CCCCGGGTTCGAAATCGATAAAAAG










(SEQ











ID NO:











2317)














Anneal-




Ampli-

Entero-

Strepto-
Strepto-

ing




con
Entero-
coccus
Listeria
coccus
coccus
Strepto-
temper-



Sequence
size
coccus
galli-
monocy-
agala-
pneu
coccus
ature


Primer
(all 25 nucleotides)
(bp)
faecium
narum
togenes
ctiae
moniae
pyogenes
(° C.)





SEQ ID
ATCAAAAAGTTGGCGAACCTTTTCA
125
−
−
−
−
−
−
55


NO: 145











SEQ ID
CAAAAGAGCGTGGAGAAAAGTATCA










NO: 146











 


VBsep3
CATAGTCTGATTGCTCAAAGTCTTG
208
−
−
−
−
−
−
55


(SEQ











ID NO:











2308)











VBsep4
GCGAATAGTGAACTACATTCTGTTG

−
−
−
−
−
−
60 


(SEQ











ID NO:











2309)











 


VBsep5
CACGCTCTTTTGCAATTTCCATTGA
208
−
−
−
−
−
−
55


(SEQ











ID NO:











2310)











VBsep6
GAAGCAAATATTCAAAATGCACCAG

NT
NT
NT
NT
NT
NT
65


(SEQ











ID NO:











2311)











 


VBsep7
AAAGTCTTTTGCTTCTTCAGATTCA
177
−
−
−
−
−
−
55


(SEQ











ID NO:











2312)











VBsep8
GTGTTCACAGGTATGGATGCTCTTA

NT
NT
NT
NT
NT
NT
60


(SEQ


NT
NT
NT
NT
NT
NT
65


ID NO:











2313)











 


VBsep9
GAGCATCCATACCTGTGAACACAGA
153
−
−
−
−
−
−
55


(SEQ











ID NO:











2314)











VBsep10
TTTTCCAATTACAAGAGACATCAGT

NT
NT
NT
NT
NT
NT
60


(SEQ


NT
NT
NT
NT
NT
NT
65


ID NO:











2315)











 


VBsep11
TTTGAATTCGCATGTACTTTGTTTG
135
−
−
−
−
−
−
60 


(SEQ











ID NO:











2316)











VBsep12
CCCCGGGTTCGAAATCGATAAAAAG










(SEQ











ID NO:











2317)





1SEQ ID NO. from U.S. pat. 6.001.564.


2All PCR assays were performed with 1 ng of purified genomic DNA by using an anneaiing temperature of 55 to 65° C. and 30 cycles of amplification. The genomic DNA from the barious bacterial species above was always isolated from reference strains obtained from ATCC.


3All positive results showed a strong amplification signal with genomic DNA from the target species S. epidermidis. The instensity of the positive amplification signal with species other than S. epidermidis was variable.


NT = NOT TESTED.













TABLE 23





Influence of nucleotide variation(s) on the efficiency of the PCR amplification:


Example with SEQ ID NO: 146 from S. epidermidis.
















1All PCR tests were performed with SEQ ID NO: 145 without modification combined with SEQ ID NO: 146 or 13 modified versions of SEQ ID NO: 146. Boxed nucleotides indicate changes in SEQ ID NO: 146.


2The tests with S. epidermidis were performed by using an annealing temperature of 55° C. with 1, 0,1 and 0,01 ng of purified genomic DNA or at 50° C. with 1 ng of purified genomic DNA.


3The tests with S. aureus were performed only at 50° C. with 1 ng of genomic DNA.


4The intensity of the positive amplification signal was quantified as follows: 3+ = strong signal, 2+ = intermediate signal and + = weak signal.













TABLE 24







Effect of the primer length on the efficiency of the PCR amplification1:


Example with the AT-rich SEQ ID NO:1452 and SEQ ID NO:1462 from S. epidermidis.




















Staph-
Staph-





STAPHYLOCOCCUS

Staphyl-
ylo-
ylo-





EPIDERMIDIS3
Staphyl-
ococcus
coccus
coccus





ATCC 14990
ococcus
haemo-
cap-
warn-

















Length
45° C.
55° C.
aureus
lyticus
itis
eri























Primer
Sequence
(nt)
1
0,1
0,01
1
0,1
0,01
45
55
45
55
45
55
45
55





VBsep301
ATATCATCAAAAAGTTGGCGAACCTTTTCA
30
NT
NT
NT
4+
3+
2+
NT
−
NT
−
NT
−
NT
−


(SEQ


















ID NO:


















2331)


















VBsep302
AATTGCAAAAGAGCGTGGAGAAAAGTATCA
30
















(SEQ


















ID NO:


















2332)


















 


SEQ ID
     ATCAAAAAGTTGGCGAACCTTTTCA
25
4+5
3+
2+
4+
3+
2+
−
−
−
−
+





NO:145


















(SEQ


















ID NO:


















2333)


















SEQ ID
     CAAAAGAGCGTGGAGAAAAGTATCA
25
















NO:146


















(SEQ


















ID NO:


















2334)


















 


VBsep201
          AAAGTTGGCGAACCTTTTCA
20
NT
NT
NT
4+
3+
2+
NT
−
NT
−
NT
−
NT
−


(SEQ


















ID NO:


















2335


















VBsep202
          GAGCGTGGAGAAAAGTATCA
20
















(SEQ


















ID NO:


















2336)


















 


VBsep171
             GTTGGCGAACCTTTTCA
17
4+
3+
2+
3+
2+
+
−
−
−
−
−
−
−
−


(SEQ


















ID NO:


















2337)


















VBsep172
             CGTGGAGAAAAGTATCA
17
















(SEQ


















ID NO:


















2338)


















 


VBsep151
               TGGCGAACCTTTTCA
15
3+
2+
+
−
−
−
−
−
−
−
−
−
−
−


(SEQ


















ID NO:


















2339)


















VBsep152
               TGGAGAAAAGTATCA
15
















(SEQ


















ID NO:


















2340





1All PCR tests were performed using an annealing temperature of 45 or 55° C. and 30 cycles of amplification.


2All SEQ ID NOs. in this Table are from U.S. pat. 6001,546.


3The tests with S. epidermidis were made with 1, 0,1 and 0,01 ng of purified genomic DNA.


4The tests with all other bacterial species were made only with 1 ng of purified genomic DNA.


5The intensity of the positive amplification signal was quantified as follows: 4+ = very strong signal, 3+ = strong signal, 2+ = intermediate signal and + = weak signal.


NT = not tested.













TABLE 25







Effect of the primer length on the efficiency of the PCR amplification1:


Example with the GC-rich SEQ ID NO:832 and SEQ ID NO:842 from P. aeruginosa.























Steno-







PSEUDOMONAS
Pseuod-


otrop-

Haemo-





AERUGINOSA3
monas
Burk-
Shew
homonas
Neisseria
philus




length
ATCC 35554
fluore-
holderia
nella
matto-
mening-
parahae-


















Primer
Sequence
(nt)
1
0,1 
0,01
scens4
cepacia
putida
philia
itidis
molyticus





SEQ ID
CGAGCGGGTGGTGTTCATC
19 
2+5
+
−
−
−
−
−
−
−


NO: 83













SEQ ID 
CAAGTCGTCGTCGGAGGGA
19











NO: 84













 


Pse554-16a
CGAGCGGGTGGTGTTC
16
2+
+
−
−
−
−
−
−
−


(SEQ













ID NO:













2341)













Pse674-16a
GTCGTCGTCGGAGGGA
16











(SEQ













ID NO:













2342)













 


Pse554-13b
GCGGGTGGTGTTC
13
2+
+
−
−
−
−
−
−
−


(SEQ













ID NO:













2343)













Pse674-13a
GTCGTCGGAGGGA 13












(SEQ













ID NO:













2344)





1All PCR tests were performed using an annealing temperature of 55° C. and 30 cycles of amplification.


2All SEQ ID NOs. in this Table are fmm U.S. pat. 6,001,546.


3The tests with P. aeruginosa were made with 1, 0,1 and 0,01 ng of purified genomic DNA.


4The tests with all other bacterial species were made only with 1 ng of purified genomic DNA.


5The intensity of the positive amplification signal was quantified as follows: 2+ = strong signal and + = moderately strong signal.













TABLE 39







Specific and ubiquitous primers for nucleic acid amplification (tuf sequences).











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position













Bacterial species:
Acinetobacter baumannii




1692
5′-GGT GAG AAC TGT GGT ATC TTA CTT
   1
478-501


1693a
5′-CAT TTC AAC GCC TTC TTT CAA CTG
   1
691-714


 


Bacterial species:
Chlamydia pneumoniae




 630
5′-CGG AGC TAT CCT AGT CGT TTC A
  20
 2-23


 629a
5′-AAG TTC CAT CTC AAC AAG GTC AAT A
  20
146-170


 


2085
5′-CAA ACT AAA GAA CAT ATC TTG CTA
  20
45-68


2086a
5′-ATA TAA TTT GCA TCA CCT TCA AG
  20
237-259


 


2087
5′-TCA GCT CGT GGG ATT AGG AGA G
  20
431-452


2088a
5′-AGG CTT CAC GCT GTT AGC CTG A
  20
584-605


 


Bacterial species:
Chlamydia trachomatis




 554
5′-GTT CCT TAC ATC GTT GTT TTT CTC
  22
 82-105


 555a
5′-TCT CGA ACT TTC TCT ATG TAT CCA
  22
249-272


 


Parasitical species:
Cryptosporidium parvum




 798
5′-TGG TTG TCC GAG CCG ATC GTT T
 865
158-179


 804a
5′-CCT GGG ACG GCC TCT GGC AT
 865
664-683


 


 799
5′-ACC TGT GAA TAC AAG CAA TCT
 865
280-300


 805a
5′-CTC TTG TCC ATC TTA GCA GT
 865
895-914


 


 800
5′-GAT GAA ATC TTC AAC GAA GTT GAT
 865
307-330


 806a
5′-AGC ATC ACC AGA CTT GAT AAG
 865
946-966


 


 801
5′-ACA ACA CCG AGA AGA TCC CA
 865
353-372


 803a
5′-ACT TCA GTG GTA ACA CCA GC
 865
616-635


 


 802
5′-TTG CCA TTT CTG GTT TCG TT
 865
377-396


 807A
5′-AAA GTG GCT TCA AAG GTT GC
 865
 981-1000


 


Bacterial species:
Enterococcus faecium




1696
5′-ATG TTC CTC TAG TTG CTG GA
  64
189-208


1697a
5′-TTT CTT CAG CAA TAC CAA CAA C
  64
422-443


 


Bacterial species:
Klebsiella pneumoniae




1329
5′-TGT AGA GCG CCG TAT CAT CAA AGT A
 103
352-377


1330a
5′-AGA TTC GAA CTT GCT GTG CGG G
 103
559-571







aThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.













Bacterial species:
Mycoplasma pneumoniae




2093
5′-TGT TGG CAA TCG AAG ACA CC
2097a
635-654


2094a
5′-TTC AAT TTC TTG ACC TAC TTT CAA
2097a
709-732


 


Bacterial species:
Neisseria gonorrhoeae




 551
5′-GAA GAA AAA ATC TTC GAA CTG GCT A
 126
256-280


 552b
5′-TAC ACG GCC GGT GAC TAC G
 126
378-396


 


2173
5′-AAG AAA AAA TCT TCG AAC TGG CTA
 126
257-280


2174b
5′-TCT ACA CGG CCG GTG
 126
384-398


 


2175
5′-CCG CCA TAC CCC GTT T
 126
654-669


2176b
5′-CGG CAT TAC CAT TTC CAC ACC TTT
 126
736-759


 


Bacterial species:
Pseudomonas aeruginosa




1694
5′-AAG GCA AGG ATG ACA ACG GC
 153
231-250


1695b
5′-ACG ATT TCC ACT TCT TCC TGG
 153
418-438


 


Bacterial species:
Streptococcus agalactiae




 549
5′-GAA CGT GAT ACT GAC AAA CCT TTA
207-210c
308331d


 550b
5′-GAA GAA GAA CAC CAA CGT TG
207-210c
520539d


 


Bacterial species:
Streptococcus pyogenes




 999
5′-TTG ACC TTG TTG ATG ACG AAG AG
1002
143-165


1000b
5′-TTA GTG TGT GGG TTG ATT GAA CT
1002
622-644


 


1001
5′-AAG AGT TGC TTG AAT TAG TTG AG
1002
161-183


1000b
5′-TTA GTG TGT GGG TTG ATT GAA CT
1002
622-644


 


Parasitical species:
Trypanosoma brucei




 820
5′-GAA GGA GGT GTC TGC TTA CAC
 864
513-533


 821b
5′-GGC GCA AAC GTC ACC ACA TCA
 864
789-809


 


 820
5′-GAA GGA GGT GTC TGC TTA CAC
 864
513-533


 822b
5′-CGG CGG ATG TCC TTA ACA GAA
 864
909-929







aSequence from databases.


bThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


cThese sequences were aligned to derive the corresponding primer.


dThe nucleotide positions refer to the S. agalactiae tuf sequence fragment


(SEQ ID NO. 209)













Parasitical species:
Trypanosoma cruzi




 794
5′-GAC GAC AAG TCG GTG AAC TT
840-842a
 281-300c


 795b
5′-ACT TGC ACG CGA TGT GGC AG
840-842a
 874-893c


 


Bacterial genus:
Clostridium sp.




 796
5′-GGT CCA ATG CCW CAA ACW AGA
32, 719-





724, 736a



 797b
5′-CAT TAA GAA TGG YTT ATC TGT SKC TCT
32, 719-
 320-346d




724, 736a



 


 808
5′-GCI TTA IWR GCA TTA GAA RAY CCA
32, 719-
 224-247d




724, 736a



 809b
5′-TCT TCC TGT WGC AAC TGT TCC TCT
32,719-
 337-360d




724, 736a



 


 810
5′-AGA GMW ACA GAT AAR SCA TTC TTA
32, 719-
 320-343d




724, 736a



 811b
5′-TRA ART AGA ATT GTG GTC TRT ATC C
32, 719-
 686-710d




724, 736a



 


Bacterial genus:
Corynebacterium sp.




 545
5′-TAC ATC CTB GTY GCI CTI AAC AAG TG
34-44, 662a
  89-114e


 546b
5′-CCR CGI CCG GTR ATG GTG AAG AT
34-44, 662a
 350-372e


 


Bacterial genus:
Enterococcus sp.




 656
5′-AAT TAA TGG CTG CAG TTG AYG A
58-72a
 273-294f


 657b
5′-TTG TCC ACG TTC GAT RTC TTC A
58-72a
 556-577f


 


 656
5′-ART TAA TGG CTG CAG TTG AYG A
58-72a
 273-294f


 271b
5′-TTG TCC ACG TTG GAT RTC TTC A
58-72a
 556-577f


 


1137
5′-ART TAA TGG CTG CWG TTG AYG AR
58-72a
 273-295f


1136b
5-ACT TGT CCA CGT TSG ATR TCT
58-72a
 559-579f


 







aThese sequences were aligned to derive the corresponding primer.


bThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


cThe nucleotide positions refer to the T. cruzi tuf sequence fragment


(SEQ ID NO. 842)


dThe nucleotide positions refer to the C. perifringens tuf sequence fragment


(SEQ ID NO. 32).


eThe nucleotide positions refer to the C. diphtheriae tuf sequence fragment


(SEQ ID NO. 662)


fThe nucleotide positions refer to the E. durans tuf sequence fragment


(SEQ ID NO. 61).













Bacterial genus:
Legionella sp.




2081
5′-GRA TYR TYA AAG TTG GTG AGC AAG
111-112a
 411-434b


2O82c
5′-CMA CTT CAT CYC GCT TCG TAC C
111-112a
 548-569b


 


Bacterial genus:
Staphylococcus sp.




 553
5′-GGC CGT GTT GAA CGT GGT CAA ATC A
176-203a
 313-337d


 707c
5′-TIA CCA TTT CAG TAC CTT CTG GTA A
176-203a
 653-677d


 


 553
5′-GGC CGT GTT GAA CGT GGT CAA ATC A
176-203a
 313-337d


 707c
5′-TWA CCA TTT CAG TAC CTT CTG GTA A
176-203a
 653-677d


 


Bacterial genus:
Streptococcus sp.




 547
5′-GTA CAG TTG CTT CAG GAC GTA TC
206-231a
 372-394e


 548c
5′-ACG TTC GAT TTC ATC ACG TTG
206-231a
 548-568e


 


Fungal genus:
Candida sp.




 576
5′-AAC TTC RTC AAG AAG GTY GCT TAC AA
407-426,
 332-357f




428-432a



 632c
5′-CCC TTT GGT GGR TCS TKC TTG GA
407-426,
 791-813f




428-432a



 


 631
5′-CAG ACC AAC YGA IAA RCC ATT RAG AT
407-426,
523-548f




428-432a



632c
5′-CCC TTT GGT GGR TCS TKC TTG GA
407-426,
791-513f




428-432a



 


 633
5′-CAG ACC AAC YGA IAA RCC ITT RAG AT
407-426,
 523-548f




428-432a



 


 632c
5′-CCC TTT GGT GGR TCS TKC TTG GA
407-426,
 791-813f




428-432a








aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the L. pneumophila tuf sequence fragment


(SEQ ID NO. 112)


cThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


dThe nucleotide positions refer to the S. aureus tuf sequence fragment


(SEQ ID NO. 179)


eThe nucleotide positions refer to the S. agalactiae tuf sequence fragment


(SEQ ID NO. 209).


fThe nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment


(SEQ ID NO. 408)













Fungal genus:
Cryptococcus sp.




1971
5′-CYG ACT GYG CCA TCC TYA TCA
434, 623, 1281,
 150-170b




1985, 1986a



1973c
5′-RAC ACC RGI YTT GGW ITC CTT
434, 623, 1281,
 464-484b




1985, 1986a



 


1972
5′-MGI CAG CTC ATY ITT GCW KSC
434, 623, 1281,
 260-280b




1985, 1986a



1973c
5′-RAC ACC RGI YTT GGW ITC CTT
434, 623, 1281,
 464-484b


1985, 1986a





 


Parasitical genus:
Entamoeba sp.




 703
5′-TAT GGA AAT TCG AAA CAT CT
512
38-57


 704c
5′-AGT GCT CCA ATT AAT GTT GG
512
442-461


 


 703
5′-TAT GGA AAT TCG AAA CAT CT
512
38-57


 705c
5′-GTA CAG TTC CAA TAC CTG AA
512
534-553


 


 703
5′-TAT GGA AAT TCG AAA CAT CT
512
38-57


 706c
5′-TGA AAT CTT CAC ATC CAA CA
512
768-787


 


 793
5′-TTA TTG TTG CTG CTG GTA CT
512
149-168


 704c
5′-AGT GCT CCA ATT AAT GTT GG
512
442-461


 


Parasitical genus:
Giardia sp.




 816
5′-GCT ACG ACG AGA TCA AGG GC
513
305-324


 819c
5′-TCG AGC TTC TGG AGG AAG AG
513
895-914


 


817
5′-TGG AAG AAG GCC GAG GAG TT
513
355-374


818c
5′-AGC CGG GCT GGA TCT TCT TC
513
825-844


 


Parasitical genus:
Leishmania sp.




 701
5′-GTG TTC ACG ATC ATC GAT GCG
514-526a
 94-114d


 702c
5′-CTC TCG ATA TCC GCG AAG CG
514-526a
  913-932d







aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the C. neoformans tuf (EF-1) sequence


fragment (SEQ ID NO. 623).


cThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


dThe nucleotide positions refer to the L. tropica tuf(EF-1) sequence fragment


(SEQ ID NO. 526)













Parasitical genus:
Trypanosoma sp.




 823
5′-GAG CGG TAT GAY GAG ATT GT
529, 840-
 493-512b




842, 864a



 824c
5′-GGC TTC TGC GGC ACC ATG CG
529, 840-
 1171-1190b




842, 864b



 


Bacterial family:
Enterobacteriaceae




 933
5′-CAT CAT CGT ITT CMT GAA CAA RTG
78, 103, 146,
 390-413d




168, 238, 698a



 934c
5′-TCA CGY TTR RTA CCA CGC AGI AGA
78, 103, 146,
 831-854d




168, 238, 698a



 


Bacterial family:
Mycobacteriaceae




 539
5′-CCI TAC ATC CTB GTY GCI CTI AAC AAG
122
 85-111


 540c
5′-GGD GCI TCY TCR TCG WAI TCC TG
122
181-203


 


Bacterial group:
Escherichia coli and Shigella




1661
5′-TGG GAA GCG AAA ATC CTG
1668e
283-300


1665c
5′-CAG TAG AGG TAG ACT TCT G
1668e
484-502


 


Bacterial group:
Pseudomonads group




 541
5′-GTK GAA ATG TTC CGC AAG CTG CT
153-155a
 476-498f


 542c
5′-CGG AAR TAG AAC TGS GGA CGG TAG
153-155a
 679-702f


 


 541
5′-GTK GAA ATG TTC CGC AAG CTG CT
153-155a
 476-498f


 544c
5′-AYG TTG TCG CCM GGC ATT MCC AT
153-155a
 749-771f







aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the T. brucei tuf (EF-1) sequence fragment


(SEQ ID NO. 864)


cThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


dThe nucleotide positions refer to the E. coli tuf sequence fragment


(SEQ ID NO. 698)


eSequence from databases.


fThe nucleotide positions refer to the P. aeruginosa tuf sequence fragment


(SEQ ID NO. 153)













Parasitical group:
Trypanosomatidae family




 923
5′-GAC GCI GCC ATC CTG ATG ATC
511, 514-526,
 166-188b




529, 840-842,





864a



 924
5′-ACC TCA GTC GTC ACG TTG GCG
511, 514-526,
 648-668b




529, 840-842,





864a



 


 925
5′-AAG CAG ATG GTT GTG TGC TG
511, 514-526,
 274-293b




529, 840-842,





864a



 926c
5′-CAG CTG CTC GTG GTG CAT CTC GAT
511, 514-526,
 676-699b




529, 840-842,





864a



 


 927
5′-ACG CGG AGA AGG TGC GCT T
511,514-526,
 389-407b




529, 840-842,





864a



 928c
5′-GGT CGT TCT TCG AGT CAC CGC A
511, 514-526,
 778-799 b




529, 840-842,





864a



 



Universal primers (bacteria)




 636
5′-ACT GGY GTT GAI ATG TTC CGY AA
7, 54, 78,
 470-492d




100, 103, 159,





209, 224, 227b



 637c
5′-ACG TCA GTI GTA CGG AAR TAG AA
7, 54, 78,
 692-714d




100, 103, 159,





209, 224, 227b



 


 638
5′-CCA ATG CCA CAA ACI CGT GAR CAC AT
7, 54, 78,
 35-60e




100, 103, 159,





209, 224, 227b



 639c
5′-TTT ACG GAA CAT TTC WAC ACC WGT IAC A
7, 54, 78,
 469-496e




100, 103, 159,





209, 224, 227b








aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the L. tropica tuf (EF-1) sequence fragment


(SEQ ID NO. 526)


cThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


dThe nucleotide positions refer to the E. coli tuf sequence fragment


(SEQ ID NO. 78).


eThe nucleotide positions refer to the B. cereus tuf sequence fragment


(SEQ ID NO. 7).













 643
5′-ACT GGI GTI GAR ATG TTC CGY AA
1, 3, 4, 7, 12,
 470-492b




13, 16, 49, 54,





72, 78, 85, 88,





91, 94, 98, 103,





108, 112, 115,





116, 120, 121,





126, 128, 134,





136, 146, 154,





159, 179, 186,





205, 209, 212,





224, 238a



 644c
5′-ACG TCI GTI GTI CKG AAR TAG AA
same as SEQ
 692-714b




ID NO. 643



 


 643
5′-ACT GGI GTI GAR ATG TTC CGY AA
1, 3, 4, 7, 12,
 470-492b


13, 16, 49, 54,







72, 78, 85, 88,





91, 94, 98, 103,





108, 112, 115,





116, 120, 121,





126, 128, 134,





136, 146, 154,





159, 179, 186,





205, 209, 212,





224, 238a



 645c
5′-ACG TCI GTI GTI CKG AAR TAR AA
same as SEQ
692-714b




ID NO. 643



 


 646
5′-ATC GAC AAG CCI TTC YTI ATG SC
2, 13, 82
 317-339d




122, 145a



 647c
5′-ACG TCC GTS GTR CGG AAG TAG AAC TG
2, 13, 82
686-711d




122, 145a



 


 646
5′-ATC GAC AAG CCI TTC YTI ATG SC
2, 13, 82
 317-339d




122, 145a



 648c
5′-ACG TCS GTS GTR CGG AAG TAG ARC TG
2, 13, 82
 686-711d




122, 145a








aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the E. coil tuf sequence fragment


(SEQ ID NO. 78).


cThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


dThe nucleotide positions refer to the A. meyeri tuf sequence fragment


(SEQ ID NO. 2)













 649
5′-GTC CTA TGC CTC ARA CWC GIG AGC AC
8, 86, 141, 143a
 33-58b


 650c
5-TTA CGG AAC ATY TCA ACA CCI GT
8, 86, 141, 143a
 473-495b


 


 636
5′-ACT GGY GTT GAl ATG TTC CGY AA
8, 86, 141, 143a
473-495b


 651c
5′-TGA CGA CCA CCI TCY TCY TTY TTC A
8, 86, 141, 143a
639-663b


 



Universal primers (fungi)




1974
5′-ACA AGG GIT GGR MSA AGG AGA C
404, 405, 433,
 443-464d




445, 898, 1268,





1276, 1986a



1975c
5′-TGR CCR GGG TGG TTR AGG ACG
404, 405, 433,
 846-866d




445, 898, 1268,





1276, 1986a



 


1976
5′-GAT GGA YTC YGT YAA ITG GGA
407-412,
 286-306e




414-426, 428-





431, 439, 443, 447,





448, 622, 624, 665,





1685, 1987-1990a



1977
5′CAT CIT GYA ATG GYA ATC TYA AT
same as SEQ
 553-575e




ID NO. 1976



 


1977
5′-GAT GGA YTC YGT YAA RTG GGA
same as SEQ
286-306e




ID NO. 1976



1979c
5-CAT CYT GYA ATG GYA ASC TYA AT
same as SEQ
 553-575e




ID NO. 1976



 


1981
5′-TGG ACA CCI SCA AGI GGK CYG
401-405,
 281-301d




433, 435, 436,





438, 444, 445, 449,





453, 455, 457, 779,





781-783, 785, 786,





788-790, 897-903,





1267-1272, 1274-1280,





1282-1287, 1991-1998a



1980c
5′-TCR ATG GCI TCI AIR AGR GTY T
same as SEQ
 488-509d




ID NO. 1981








aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the B. distasonis tuf sequence fragment


(SEQ ID NO. 8)


cThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


dThe nucleotide positions refer to the A. fumigatus tuf (EF-1) sequence


fragment (SEQ ID NO. 404)


eThe nucleotide positions refer to the C. albicans tuf (EF-1) sequence fragment


(SEQ ID NO. 407)













1982
5′-TGG ACA CYI SCA AGI GGK CYG
same as SEQ
 281-301a




ID NO. 1981



1980b
5′-TCR ATG GCI TCI AIR AGR GTY T
same as SEQ
 488-509a




ID NO. 1981



 


1983
5′-CYG AYT GCG CYA TIC TCA TCA
same as SEQ
143-163a




ID NO. 1981



1980b
5′-TCR ATG GCI TCI AIR AGR GTY T
same as SEQ
488-509a




ID NO. 1981



 


1984
5′-CYG AYT GYG CYA TYC TSA TCA
same as SEQ
 143-163a




ID NO. 1981



1980b
5′-TCR ATG GCI TCI AIR AGR GTY T
same as SEQ
 488-509a




ID NO. 1981



 



Sequencing primers




 556
5′-CGG CGC NAT CYT SGT TGT TGC
668c
306-326


 557b
5′-CCM AGG CAT RAC CAT CTC GGT G
668c
1047-1068


 


 694
5′-CGG CGC IAT CYT SGT TGT TGC
668c
306-326


 557b
5′-CCM AGG CAT RAC CAT CTC GGT G
668c
1047-1068


 


 664
5′-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA
619c
604-632


 652b
5′-CCW AYA GTI YKI CCI CCY TCY CTI ATA
619c
1482-1508


 


 664
5′-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA
619c
604-632


 561b
5′-ACI GTI CGG CCR CCC TCA CGG AT
619c
1483-1505


 


 543
5′-ATC TTA GTA GTT TCT GCT GCT GA
607
 8-30


 660b
5′-GTA GAA TTG AGG ACG GTA GTT AG
607
678-700


 


 658
5′-GAT YTA GTC GAT GAT GAA GAA TT
621
116-138


 659b
5′-GCT TTT TGI GTT TCW GGT TTR AT
621
443-465


 


 658
5′-GAT YTA GTC GAT GAT GAA GAA TT
621
116-138


 661b
5′-GTA GAA YTG TGG WCG ATA RTT RT
621
678-700


 


 558
5′-TCI TTY AAR TAY GCI TGG GT
665c
157-176


 559b
5′-CCG ACR GCR AYI GTY TGI CKC AT
665c
1279-1301


 


 813
5′-AAT CYG TYG AAA TGC AYC ACG A
665c
687-708


 559b
5′-CCG ACR GCR AYI GTY TGI CKC AT
665c
1279-1301







aThe nucleotide positions refer to the A. fumigatus tuf (EF-1) sequence


fragment (SEQ ID NO. 404).


bThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


cSequences from databases.













 558
5′-TCI TTY AAR TAY GCI TGG GT
665a
157-176


 815b
5′-TGG TGC ATY TCK ACR GAC TT
665a
686-705


 


 560
5′-GAY TTC ATY AAR AAY ATG ATY AC
665a
289-311


 559b
5′-CCG ACR GCR AYI GTY TGI CKC AT
665a
1279-1301


 


 653
5′-GAY TTC ATI AAR AAY ATG AT
665a
289-308


 559b
5′-CCG ACR GCR AYI GTY TGI CKC AT
665a
1279-1301


 


 558
5′-TCI TTY AAR TAY GCI TGG GT
665a
157-176


 655b
5′-CCR ATA CCI CMR ATY TTG TA
665a
754-773


 


 654
5′-TAC AAR ATY KGI GGT ATY GG
665a
754-773


 559b
5′-CCG ACR GCR AYI GTY TGI CKC AT
665a
1279-1301


 


 696
5′-ATI GGI CAY RTI GAY CAY GGI AAR AC
698a
52-77


 697b
5′-CCI ACI GTI CKI CCR CCY TCR CG
698a
1132-1154


 


 911
5′-GAC GGM KKC ATG CCG CAR AC
 853
22-41


 914b
5′-GAA RAG CTG CGG RCG RTA GTG
 853
700-720


 


 912
5′-GAC GGC GKC ATG CCG CAR AC
 846
20-39


 914b
5′-GAA RAG CTG CGG RCG RTA GTG
 846
692-712


 


 913
5′-GAC GGY SYC ATG CCK CAG AC
 843
251-270


 915b
5′-AAA CGC CTG AGG RCG GTA GTT
 843
905-925


 


 916
5′-GCC GAG CTG GCC GGC TTC AG
 846
422-441


 561b
5′-ACI GTI CGG CCR CCC TCA CGG AT
619a
1483-1505


 


 664
5′-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA
619a
604-632


 917b
5′-TCG TGC TAC CCG TYG CCG CCA T
 846
593-614







aSequences from databases.


bThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.













1221
5′-GAY ACI CCI GGI CAY GTI GAY TT
1230a
292-314


1226b
5′-GTI RMR TAI CCR AAC ATY TC
1230a
2014-2033


 


1222
5′-ATY GAY ACI CCI GGI CAY GTI GAY TT
1230a
289-314


1223b
5′-AYI TCI ARR TGI ARY TCR CCC ATI CC
1230a
1408-1433


 


1224
5′-CCI GYI HTI YTI GAR CCI ATI ATG
1230a
1858-1881


1225b
5′-TAI CCR AAC ATY TCI SMI ARI GGI AC
1230a
2002-2027


 


1227
5′-GTI CCI YTI KCI GAR ATG TTY GGI TA
1230a
2002-2027


1229b
5′-TCC ATY TGI GCI GCI CCI GTI ATC AT
 698a
 4-29


 


1228
5′-GTI CCI YTI KCI GAR ATG TTY GGI TAY GC
1230a
2002-2030


1229b
5′-TCC ATY TGI GCI GCI CCI GTI ATC AT
 698a
 4-29


 


1999
5′-CAT GTC AAY ATT GGT ACT ATT GGT CAT GT 498-500,
 25-53d





502, 505, 506,





508, 619, 2004, 2005c



2000b
5′-CCA CCY TCI CTC AMG TTG AAR CGT T
same as SEQ
1133-1157d




ID NO. 1999



 


2001
5′-ACY ACI TTR ACI GCY GCY ATY AC
same as SEQ
 67-89d




ID NO. 1999



2003b
5′-CAT YTC RAI RTT GTC ACC TGG
same as SEQ
1072-1092d




ID NO. 1999



 


2002
5′-CCI GAR GAR AGA GCI MGW GGT
same as SEQ
151-171d




ID NO. 1999



2003b
5′-CAT YTC RAI RTT GTC ACC TGG
same as SEQ
1072-1092d




ID NO. 1999





aSequences from databases.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.


cThese sequences were aligned to derive the corresponding primer.


dThe nucleotide positions refer to the C. albicans tuf sequence fragment (SEQ ID NO. 2004)













TABLE 40







Specific and ubiquitous primers for nucleic acid amplification (atpD sequences).











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Bacterial species:
Acinetobacter baumannii




1690
5′-CAG GTC CTG TTG CGA CTG AAG AA
 243
186-208


1691b
5′-CAC AGA TAA ACC TGA GTG TGC TTT C
 243
394-418


 


Bacterial species:
Bacteroides fragilis




2134
5′-CGC GTG AAG CTT CTG TG
 929
184-200


2351b
5′-TCT CGC CGT TAT TCA GTT TC
 929
395-414


 


Bacterial species:
Bordetella pertussis




2180
5′-TTC GCC GGC GTG GGC
1672c
544-558


2181b
5′-AGC GCC ACG CGC AGG
1672c
666-680


 


Bacterial species:
Enterococcus faecium




1698
5′-GGA ATC AAC AGA TGG TTT ACA AA
 292
131-153


1699b
5′-GCA TCT TCT GGG AAA GGT GT
 292
258-277


 


1700
5′-AAG ATG CGG AAA GAA GCG AA
 292
271-290


1701b
5′-ATT ATG GAT CAG TTC TTG GAT CA
 292
439-461


 


Bacterial species:
Klebsiella pneumoniae




1331
5′-GCC CTT GAG GTA CAG AAT GGT AAT GAA GTT
 317
88-118


1332b
5′-GAC CGC GGC GCA GAC CAT CA
 317
183-203


 







aThese sequences were aligned to derive the corresponding primer.


bThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


cSequence from databases.













Bacterial species:
Streptococcus agalactiae




 627
5′-ATT GTC TAT AAA AAT GGC GAT AAG TC
379-383a
52-67b


 625c
5′-CGT TGA AGA CAC GAC CCA AAG TAT CC
379-383a
206-231b


 


 628
5′-AAA ATG GCG ATA AGT CAC AAA AAG TA
379-383a
52-77b


 625c
5′CGT TGA AGA CAC GAC CCA AAG TAT CC
379-383a
206-231b


 


 627
5′-ATT GTC TAT AAA AAT GGC GAT AAG TC
379-383a
42-67b


 626b
5′-TAC CAC CTT TTA AGT AAG GTG CTA AT
379-383a
371-396b


 


 628
5′-AAA ATG GCG ATA AGT CAC AAA AAG TA
379-383a
52-77b


 626c
5′-TAC CAC CTT TTA AGT AAG GTG CTA AT
379-383a
371-396b


 


Bacterial group:
Campylobacter jejuni and C. coli




2131
5′-AAG CMA TTG TTG TAA ATT TTG AAA G
1576, 1600,
7-31e




1849, 1863, 2139d, a



2132c
5′-TCA TAT CCA TAG CAA TAG TTC TA
1576, 1600,
92-114e




1849, 1863, 2139d, a



 


Bacterial genus:
Bordetella sp.




 825
5′-ATG AGC ARC GSA ACC ATC GTT CAG TG
1672d
1-26


 826c
5′-TCG ATC GTG CCG ACC ATG TAG AAC GC
1672d
1342-1367


 


Fungal genus:
Candida sp.




 634
5′-AAC ACY GTC AGR RCI ATT GCY ATG GA
460-472,
101-126f




474-478a



 635c
5′-AAA CCR GTI ARR GCR ACT CTI GCT CT
460-472, 617-642f





474-478a








aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the S. agalactiae atpD sequence fragment


(SEQ ID NO. 380)


cThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


dSequence from databases.


eThe nucleotide positions refer to the C. jejuni atpD sequence fragment


(SEQ ID NO. 1576)


fThe nucleotide positions refer to the C. albicans atpD sequence fragment


(SEQ ID NO. 460)














Universal primers




 562
5′-CAR ATG RAY GAR CCI CCI GGI GYI MGI ATG
243, 244, 262,
528-557 b




264, 280, 284,





291, 297, 309,





311, 315, 317,





324, 329, 332,





334-336, 339,





342, 343, 351,





356, 357, 364-





366, 370, 375,





379, 393a



 563c
5′-GGY TGR TAI CCI ACI GCI GAl GGC AT
243, 244, 262,
687-712b




264, 280, 284,





291, 297, 309,





311, 315, 317,





324, 329, 332,





334-336, 339,





342, 343, 351,





356, 357, 364-





366, 370, 375,





379, 393a



 


 564
5′-TAY GGI CAR ATG AAY GAR CCI CCI GGI AA
243, 244, 262,
522-550b




264, 280, 284,





291, 297, 309,





311, 315, 317,





324, 329, 332,





334-336, 339,





342, 343, 351,





356, 357, 364-





366, 370, 375,





379, 393a



 565c
5′-GGY TGR TAI CCI ACI GCI GAl GGD AT
243, 244, 262,
687-712b




264, 280, 284,





291, 297, 309,





311, 315, 317,





324, 329, 332,





334-336, 339,





342, 343, 351,





356, 357, 364-





366, 370, 375,





379, 393a








aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the K. pneumoniae atpD sequence fragment


(SEQ ID NO. 317)


cThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.













 640
5′-TCC ATG GTI TWY GGI CAR ATG AA
248, 284, 315,
513-535b




317, 343, 357,





366, 370, 379, 393a



 640c
5′-TGA TAA CCW ACI GCI GAl GGC ATA CG
248, 284, 315,
684-709b




317, 343, 357,





366, 370, 379, 393a



 


 642
5′-GGC GTI GGI GAR CGI ACI CGT GA
248, 284, 315,
438-460b




317, 343, 357,





366, 370, 379, 393a



 641c
5′-TGA TAA CCW ACI GCI GAI GGC ATA CG
248, 284, 315,
684-709b




317, 343, 357,





366, 370,379, 393a



 



Sequencing primers




 566
5′-TTY GGI GGI GCI GGI GTI GGI AAR AC
669d
445-470


 567c
5′-TCR TCI GCI GGI ACR TAI AYI GCY TG
669d
883-908


 


 566
5′-TTY GGI GGI GCI GGI GTI GGI AAR AC
669d
445-470


 814
5′-GCI GGC ACG TAC ACI GCC TG
666d
901-920


 


 568
5′-RTI ATI GGI GCI GTI RTI GAY GT
669d
25-47


 567c
5′-TCR TCI GCI GGI ACR TAI AYI GCY TG
669d
883-908


 


 570
5′-RTI RYI GGI CCI GTI RTI GAY GT
672d
31-53


 567c
5′-TCR TCI GCI GGI ACR TAI AYI GCY TG
669d
883-908


 


 572
5′-RTI RTI GGI SCI GTI RTI GA
669d
25-44


 567c
5′-TCR TCI GCI GGI ACR TAI AYI GCY TG
669d
883-908


 


 569
5′-RTI RTI GGI SCI GTI RTI GAT AT
671d
31-53


 567c
5-TCR TCI GCI GGI ACR TAI AYI GCY TG
669d
883-908


 


 571
5′-RTI RTI GGI CCI GTI RTI GAT GT
670d
31-53


 567c
5′-TCR TCI GCI GGI ACR TAI AYI GCY TG
669d
883-908







aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the K. pneumnoniae atpD sequence fragment


(SEQ ID NO. 317).


cThese sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


dSequences from databases.













 700
5′-TIR TIG AYG TCG ART TCC CTC ARG
669a
38-61


 567b
5′-TCR TCI GCI GGI ACR TAI AYI GCY TG
669a
883-908


 


 568
5′-RTI ATI GGI GCI GTI RTI GAY GT
669a
25-47


 573b
5′-CCI CCI ACC ATR TAR AAI GC
666a
1465-1484


 


 574
5′-ATI GCI ATG GAY GGI ACI GAR GG
666a
283-305


 573b
5′-CCI CCI ACC ATR TAR AAI GC
666a
1465-1484


 


 574
5′-ATI GCI ATG GAY GGI ACI GAR GG
666a
283-305


 708b
5′-TCR TCC ATI CCI ARI ATI GCI ATI AT
666a
1258-1283


 


 681
5′-GGI SSI TTY GGI ISI GGI AAR AC
685
694-716


 682b
5′-GTI ACI GGY TCY TCR AAR TTI CCI CC
686
1177-1202


 


 681
5′-GGI SSI TTY GGI ISI GGI AAR AC
685
694-716


 683b
5′-GTI ACI GGI TCI SWI AWR TCI CCI CC
685
1180-1205


 


 681
5′-GGI SSI TTY GGI ISI GGI AAR AC
685
694-716


 699
5′-GTI ACI GGY TCY TYR ARR TTI CCI CC
686
1177-1202


 


 681
5′-GGI SSI TTY GGI ISI GGI AAR AC
685
694-716


 812b
5′-GTI ACI GGI TCY TYR ARR TTI CCI CC
685
1180-1205


 


1213
5′-AAR GGI GGI ACI GCI GCI ATH CCI GG
714a
697-722


1212b
5′-CCI CCI RGI GGI GAl ACI GCW CC
714a
1189-1211


 


1203
5′-GGI GAR MGI GGI AAY GAR ATG
709a
724-744


1207b
5′-CCI TCI TCW CCI GGC ATY TC
709a
 985-1004


 


1204
5′-GCI AAY AAC ITC IWN YAT GCC
709a
822-842


1206b
5′-CKI SRI GTI GAR TCI GCC A
709a
926-944


 


1205
5′-AAY ACI TCI AWY ATG CCI GT
709a
826-845


1207b
5′-CCI TCI TCW CCI GGC ATY TC
709a
 985-1004


 


2282
5′-AGR RGC IMA RAT GTA TGA
714a
84-101


2284b
5′-TCT GWG TRA CIG GYT CKG AGA
714a
1217-1237


 


2283
5-ATI TAT GAY GGK ITT CAG AGG C
714a
271-292


2285b
5′-CMC CIC CWG GTG GWG AWA C
714a
1195-1213





aSequences from databases.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.













TABLE 41







Internal hybridization probes for specific detection of tuf sequences.











Originating DNA fragment












SEQ ID 
Nucleotide


SEQ ID NO.
Nucleotide sequence
NO.
position













Bacterial species:
Abiorophia adiacens




2170
5′-ACG TGA CGT TGA CAA ACC A
1715
313-331


 


Bacterial species:
Chlamydia pneumoniae




2089
5′-ATG CTG AAC TTA TTG ACC TT
20
136-155


2090
5′-CGT TAC TGG AGT CGA AAT G
20
467-485


 


Bacterial species:
Enterococcus faecails




 580
5′-GCT AAA CCA GCT ACA ATC ACT CCA C
62-63, 607a
584-608b


 603
5′-GGT ATT AAA GAC GAA ACA TC
62-63, 607a
440-459b


1174
5′-GAA CGT GGT GAA GTT CGC
62-63, 607a
398-415b


 


Bacterial species:
Enterococcus faecium




 602
5′-AAG TTG AAG TTG TTG GTA TT
64, 608a
426-445c


 


Bacterial species:
Enterococcus gallinarum




 604
5′-GGT GAT GAA GTA GAA ATC GT
66, 609a
419-438d


 


Bacterial species:
Escherichla coli




 579
5′-GAA GGC CGT GCT GGT GAG AA
78
503-522


2168
5′-CAT CAA AGT TGG TGA AGA AGT TG
78
409-431


 


Bacterial species:
Neisseria gonorrhoeae




2166
5′-GAC AAA CCA TTC CTG CTG
126



 


Fungal species:
Candida albicans




 577
5′-CAT GAT TGA ACC ATC CAC CA
407-411a
406-425f


 


Fungal species:
Candida dubliniensis




 578
5′-CAT GAT TGA AGC TTC CAC CA
412, 414-415a
418-437g







a These sequences were aligned to derive the corresponding primer.


b The nucleotide positions refer to the E. faecalis tuf sequence fragment (SEQ ID NO. 607)


c The nucleotide positions refer to the E. faecium tuf sequence fragment (SEQ ID NO. 608)


d The nucleotide positions refer to the E. gallinarum tuf sequence fragment (SEQ ID NO. 609)


e The nucleotide positions refer to the N. gonorrhoeae tuf sequence fragment (SEQ ID NO. 126)


f The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment


(SEQ ID NO. 408).


g The nucleotide positions refer to the C. dubliniensis tuf(EF-1) sequence fragment


(SEQ ID NO. 414).













Bacterial species:
Haemophilus Influenzae




 581
5′-ACA TCG GTG CAT TAT TAC GTG G
610a
551-572


 


Bacterial species:
Mycoplasma pneumoniae




2095
5′-CGG TCG GGT TGA ACG TGG
2097a
687-704


 


Bacterial species:
Staphylococcus aureus




 584
5′-ACA TGA CAC ATC TAA AAC AA
176-180b
 369-388c


 585
5′-ACC ACA TAC TGA ATT CAA AG
176-180b
 525-544c


 586
5′-CAG AAG TAT ACG TAT TAT CA
176-180b
 545-564c


 587
5′-CGT ATT ATC AAA AGA CGA AG
176-180b
 555-574c


 588
5′-TCT TCT CAA ACT ATC GTC CA
176-180b
 593-612c


 


Bacterial species:
Staphylococcus epidermidis




 589
5′-GCA CGA AAC TTC TAA AAC AA
185, 611b
 445-464d


 590
5′-TAT ACG TAT TAT CTA AAG AT
185, 611b
 627-646d


 591
5′-TCC TGG TTC TAT TAC ACC AC
185, 611b
 586-605d


 592
5′-CAA AGC TGA AGT ATA CGT AT
185, 611b
 616-635d


 593
5′-TTC ACT AAC TAT CGC CCA CA
185, 611b
 671-690d


 


Bacterial species:
Staphylococcus haemolyticus




 594
5′-ATT GGT ATC CAT GAC ACT TC
186, 188-190b
 437-456e


 595
5′-TTA AAG CAG ACG TAT ACG TT
186, 188-190b
 615-634e


 


Bacterial species:
Staphylococcus hominis




 596
5′-GAA ATT ATT GGT ATC AAA GA
191, 193-196b
 431-450f


 597
5′-ATT GGT ATC AAA GAA ACT TC
191, 193-196b
 437-456f


 598
5′-AAT TAC ACC TCA CAC AAA AT
191, 193-196b
 595-614f







a Sequences from databases.


b These sequences were aligned to derive the corresponding probe.


c The nucleotide positions refer to the S. aureus tuf sequence fragment (SEQ ID NO. 179)


d The nucleotide positions refer to the S. epidermidis tuf sequence fragment (SEQ ID NO. 611).


e The nucleotide positions refer to the S. haemolyticus tuf sequence fragment (SEQ ID NO. 186).


f The nucleotide positions refer to the S. hominis tuf sequence fragment (SEQ ID NO. 191)













Bacterial species:
Staphylococcus saprophyticus




 599
5′-CGG TGA AGA AAT CGA AAT CA
198-200a
 406-425b


 600
5′-ATG CAA GAA GAA TCA AGC AA
198-200a
 431-450b


 601
5′-GTT TCA CGT GAT GAT GTA CA
198-200a
 536-555b


 695
5′-GTT TCA CGT GAT GAC GTA CA
198-200a
 563-582b


 


Bacterial species:
Streptococcus agalactiae




 582c
5′-TTT CAA CTT CGT CGT TGA CAC GAA CAG T
207-210a
 404-431d


 583c
5′-CAA CTG CTT TTT GGA TAT CTT CTT TAA TAC CAA CG
207-210a
 433-467d


1199
5′-GTA TTA AAG AAG ATA TCC AAA AAG C
207-210a
 438-462d


 


Bacterial species:
Streptococcus pneumoniae




1201
5′-TCA AAG AAG AAA CTA AAA AAG CTG T
971, 977,
 513-537e




979, 986a



 


Bacterial species:
Streptococcus pyogenes




1200
5′-TCA AAG AAG AAA CTA AAA AAG CTG T
1002
473-497


 


Bacterial group:
Enterococcus casseliflavus-flavescens-





gallinarum group




 620
5′-ATT GGT GCA TTG CTA CGT
58, 65, 66a
 527-544f


1122
5′-TGG TGC ATT GCT ACG TGG
58, 65, 66a
 529-546f


 


Bacterial group:
Enterococcus sp., Gemella sp., A. adiacens




2172
5′-GTG TTG AAA TGT TCC GTA AA
58-62, 67-71,
 477-496g




87-88, 607-609,





727, 871





1715, 1722a








a These sequences were aligned to derive the corresponding primer.


b The nucleotide positions refer to the S. saprophyticus tuf sequence fragment (SEQ ID NO. 198)


c These sequences are from the complementary DNA strand of the sequence of the originating


fragment given in the Sequence Listing.


d The nucleotide positions refer to the S. agalactiae tuf sequence fragment (SEQ ID NO. 209).


e The nucleotide positions refer to the S. pneumoniae tuf sequence fragment (SEQ ID NO. 986)


f The nucleotide positions refer to the E. flavescens tuf sequence fragment (SEQ ID NO. 65)


g The nucleotide positions refer to the E. faecium tuf sequence fragment (SEQ ID NO. 608)













Bacterial genus:
Gemella




2171
5′-TCG TTG GAT TAA CTG AAG AA
87, 88a
 430-449b


 


Bacterial genus:
Staphylococcus sp.




 605
5′-GAA ATG TTC CGT AAA TTA TT
176-203a
 403-422c


 606
5′-ATT AGA CTA CGC TGA AGC TG
176-203a
 420-439c


1175
5′-GTT ACT GGT GTA GAA ATG TTC
176-203a
 391-411c


1176
5′-TAC TGG TGT AGA AAT GTT C
176-203h
 393-411c


 


Bacterial genus:
Streptococcus sp.




1202
5′-GTG TTG AAA TGT TCC GTA AAC A
206-231, 971,
 466-487d


 


Fungal species:
Candida albicans




1156
5′-GTT GAA ATG CAT CAC GAA CAA TT
407-412, 624a
 680-702e


 


Fungal group:
Candida albicans and C. tropicalis




1160
5′-CGT TTC TGT TAA AGA AAT TAG AAG
407-412,
 748-771e




429, 624a



 


Fungal species:
Candida dubliniensia




1166
5′-ACG TTA AGA ATG TTT CTG TCA A
414-415a
 750-771f


1168
5′-GAA CAA TTG GTT GAA GGT GT
414-415a
 707-726f


 


Fungal species:
Candida glabrata




1158
5′-AAG AGG TAA TGT CTG TGG T
417
781-799


1159
5′-TGA AGG TTT GCC AGG TGA
417
718-735


 


Fungal species:
Candida krusei




1161
5′-TCC AGG TGA TAA CGT TGG
422
720-737







a These sequences were aligned to derive the corresponding primer.


b The nucleotide positions refer to the C. haemolysans tuf sequence fragment (SEQ ID NO. 87)


c The nucleotide positions refer to the S. aureus tuf sequence fragment (SEQ ID NO. 179)


d The nucleotide positions refer to the S. pneuinoniae tuf sequence fragment (SEQ ID NO. 986).


e The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment (SEQ ID NO. 408)


f The nucleotide positions refer to the C. dubliniensis tuf(EF-1) sequence fragment


(SEQ ID NO. 414)













Fungal group:
Candida lusitaniae and C. guillermondii




1162
5′-CAA GTC CGT GGA AAT GCA
418, 424a
 682-699b


 


Fungal species:
Candida parapsilosis




1157
5′-AAG AAC GTT TCA GTT AAG GAA AT
426
749-771


 


Fungal species:
Candida zeylanoides




1165
5′-GGT TTC AAC GTG AAG AAC
432
713-730


 


Fungal genus:
Candida sp.




1163
5′-GTT GGT TTC AAC GTT AAG AAC
407-412, 414-
 728-748c




415, 417, 418,





422, 429a



1164
5′-GGT TTC AAC GTC AAG AAC
413, 416, 420,
 740-757b




421, 424, 425,





426, 428, 431a



1167
5′-GTT GGT TTC AAC GT
406-426, 428-
 728-741c




432, 624a





aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the C. lusitaniae tuf(EF-1) sequence fragment (SEQ ID NO. 424).


cThe nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment (SEQ ID NO. 408).













TABLE 42







Strategy for the selection of amplification/sequencing primers from atpD (F-type) sequences.












SEQ ID




23                         49   443
NO.:
Accession #:





B. cepacia
AGTgCAT CGGCGCCGTT ATCGACGTGG...TGTTCG GCGGTGCTGG CGTGGGCAAG
2346−
X76877


 


B. pertussis
AGTgCAT CGGCGCCGTG GTGGATATTC...TGTTCG GCGGCGCCGG CGTGGGCAAG
2347−
Genome project


 


P. aeruginosa
AAATCAT CGGCGCCGTG ATCGACGTGG...TGTTCG GCGGCGCCGG CGTGGGCAAG
2348−
Genome project


 


E. coli
AGGTAAT CGGCGCCGTA GTTGACGTCG...TGTTCG GTGGTGCGGG TGTAGGTAAA
−2349
J01594


 


N. gonorrhoeae
AAATTAT CGGTGCGGTT GTTGACGTGG...TGTTCG GCGGTGCCGG TGTGGGTAAA
−2350
Genome project


 


M. thermoacetica
AGGTTAT TGGCCCGGTG GTTGACGTCG...TCTTCG GCGGCGCCGG GGTCGGCAAG
−2351
U64318


 


S. aurantiaca
AGGTTcT CGGTCCCGTG ATTGACGTGG...TGTTCG GCGGCGCCGG CGTGGGCAAG
−2352
X76879


 


M. tuberculosis
GGGTCAC TGGGCCCGTC GTCGACGTCG...TGTTCG GCGGTGCCGG GGTGGGCAAG
−2353
Z73419


 


B. fragilis
AGGTAAT TGGCCCTGTG GTCGATGTGT...TGTTTG GCGGGGCCGG AGTGGGTAAA
−2354
M22247


 


C. lytica
AAATTAT TGGCCCACTT ATAGATGTGG...TATTTG GAGGTGCCGG AGTAGGTAAA
672
M22535


 


A. woodii
AGGTTAT TGGACCAGTA GTCGATGTTA...TTTTCG GTGGTGCCGG AGTTGGTAAA
−2356
U10505


 


C. acetobutyilcum
AGGTAAT AGGACCTGTT GTGGATATTA...TGTTCG GTGGTGCCGG TGTTGGTAAA
671
AF101055


 


M. pneumoniae
AAGTGAT TGGCCCGGTA GTTGATGTCA...TATTTG GTGGTGCTGG TGTTGGTAAA
−2357
U43738


 


H. pylori
AGGTTtT AGGCCCGGTG GTAGATGTGG...TGTTTG GTGGGGCTGG CGTACGCAAA
670
AF004014


 


Selected





sequences for





universal primers
  RTIAT IGGICCIGTI RTIGAYGT
568




  RTIRY IGGICCIGTI RTIGAYGT
570




  RTIRT IGGISCICTI RTIGA
572




  RTIRT IGGISCIGTI RTZGATAT
569




  RTIRT IGGICCIGTI RTIGATGT
571




                                  TTYG GIGGIGCIGG IGTIGGIAAR
566



 


Selected





sequence for





universal primera

















SEQ ID





 472   881                           910
NO.:
Accession #:






B. cepacia
ACCG...TCCA GGCCGTGT ACGTCCCTGC GGACGACT
2346−
X76877


 



B. pertussis
ACCG...TCCA GGCCGTGT ACGTGCCTGC CGACGACT
2347−
Genome project


 



P. aeruginosa
ACCG...TCCA GGCCGTAT ACGTTCCCGC GGACGACC
2348−
Genome project


 



E. coli
ACCG...TACA GGCAGTAT ACGTACCTGC GGATGACT
−2349
J01594


 



N. gonorrhoeae
ACCG...TCCA AGCCGTAT ATGTACCTGC GGATGACT
−2350
Genome project


 



M. thermoacetica
ACGG.. TGCA AGCTATCT ATGTGCCGGC CGACGACC
−2351
U64318


 



S. aurantiaca
ACGG...TGCA GGCCATCT ACGTGCCCGC CGACGACC
−2352
X76879


 



M. tuberculosis
ACGG...TGCA AGCCGTCT ACGTGCCCGC CGACGACT
−2353
Z73419


 



B. fragilis
ACGG...TGCA CGCTGTTT ACGTACCGGC TGATGACT
−2354
M22247


 



C. lytica
ACAG...TACA GGCGGTTT ACGTACCTGC GGATQATT
672
M22535


 



A. woodii
ACCG...TTCA GGCCGTTT ACGaTCCAGC CGATGACT
−2356
U10505


 



C. acetobutyilcum
ACAG...TTCA GGCTGTAT ATGTTCCTGC TGATGACC
671
AF101055


 



M. pneumoniae
ACGG...TGCA AGCGATCT ATGTGCCAGC TGATGACT
−2357
U43738


 



H. pylori
ACGG...TTCA AGCGGTGT ATGTGCCAGC AGACGACT
670
AF004014


 



Selected






sequences for






universal primers







AC
566



 



Selected






sequence for
         CA RGCIRTIT AYGTICCIGC IGAYGA
567




universal primera





The sequence numbering refers to the Escherichia coli atpD gene fragment (SEQ ID NO. 669).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lower-case letters.


Dots indicate gaps in the sequences displayed.


“R” “Y” “N” “K” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“M” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.


aThis sequence is the reverse-complement of the selected primer.













TABLE 43







Strategy for the selection of universal amplification/sequencing


primers from atpD (V-type) sequences.











SEQ ID



691                          719   1177                           1208
NO.:












E. hirae
CC AGGTCCGTTT GGTGCAGGGA AGACAGT...TCTGGTGGAg ATaTCtctGA ACCAGTGACT CA
685


 


H. salinarum
CC GGGGCCGTTC GGGTCCGGGA AGACGGT...CCCGGCGGGg ACTTCtccGA GCCGGTCACC CA
687


 


T. thermophilus
CC TGGGCCCTTC GGCAGCGGCA AGACCGT...CCGGGCGGCg ACaTgtccGA GCCCGTGACC CA
693


 


Human
CC TGGGGCCTTC GGATGTGGCA AGACTGT...CCCGGTGGAg ACTTCtcAGA tCCCGTGACG AC
688


 


T. congolense
CC TGGCGCGTTT GGATGCGGAA AGACGGT...CCTGGAGGTg ACTTTtctGA cCCAGTGACG TC
692


 


P. falciparum
CC TGGTGCATTT GGTTGTGGAA AAACTTG...CCAGGTGGTg ATTTCtctGA cCCTGTAACT AC
689


 


C. pneumoniae
CC AGGACCTTTT GGTGCAGGGA AAACAGT...GCAGGAGGAA ACTTTGAAGA ACCAGTCACT CA
686


 


Selected sequences




for universal primers
    GGISSITTY GGIISIGGIA ARAC
681


 


Selected sequences
                                      GGIGGIA AYTTYGARGA RCCIGTIAC
682


for universal primersa
                                      GGIGGIG AYWTIWSIGA ICCIGTIAC
683





The sequence numbering refers to the Enterococcus hirae atpD gene fragment (SEQ ID NO. 685).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches for SEQ ID NOs. 681 and 682 are indicated by lowercase letters.


Mismatches for SEQ ID NO. 683 are indicated by underlined nucleotides.


Dots indicate gaps in the sequences displayed.


R” “Y” “M” “K” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“M” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, C or T.


aThese sequences are the reverse-complement of the selected primers.













TABLE 44







Strategy for the selection of universal amplification/sequencing


primers from tuf (M) sequences (organelle origin).











601                                635   1479
SEQ ID NO.
Accession #:





C. neaformansa
AAGAA CATGATCACC GGTaCCtCCC AGgctGACTG...CGCcgTCcGA
−2358
U81803


 


S. cerevisiaea
AAGAA CATGATTACT GGTaCTtCTC AAgctGACTG...CGCTgTCAGA
665
X00779


 


O. volvulusa
AAGAA TATGATCACA GGTaCTtCTC AGgctGACTG...TGCTgTGcGt
−2359
M64333


 


Humana
AAAAA CATGATTACA GGGaCAtCTC AGgctGACTG...TGCTgTTcGt
−2360
X03558


 


G. max B1b
AAGAA CATGATCACC GGCGCTGCCC AGATGGACGG...TGCTATTAGA
−2361
Y15107


 


G. max B2b
AAAAA CATGATCACC GGCGCCGCCC AGATGGACGG...TGCTATTAGA
−2362
Y15108


 


E. colic
AAAAA CATGATCACC GGTGCTGCTC AGATGGACGG...CGCaATCcGt
78
—


 


S. aureofaciensc
AAGAA CATGATTCAC GGTGCCGCCC AGATGGACGG...CGCcATCcGt
−2363
AF007125


 


E. tenellab
AAGAA TATGATTACA GGAGCAGCAC AAATGGATGG...TGCTATAAGA
−2364
AI755521


 


T. gondiib
AAGAA TATGATTACT GGAGCCGCAC AAATGGATGG...TGCTATTAGA
−2365
Y11431


 


S. cerevisiaeb
AAGAA TATGATTACC GGTGCTGCTC AAATGGATGG...CAATATCAGA
619
K00428


 


A. thalianab
AAAAA TATGATTACT GGAGCTGCGC AAATGGATGG...TGCCtTAAGG
−2366
X89227


 


Selected sequence
   AA YATGATIACI GGIGCICCIC ARATGGA
664



for universal





primer





 


Selected sequences
                                             TATIAGR
652



for universal
                                            ATCCGT
561



primers
















                     1511
SEQ ID NO.
Accession #:






C. neaformansa
GAcatGcGAC AGACcGTTGc CGT
−2358
U81803


 



S. cerevisiaea
GAcatGaGAC AAACTGTcGc TGT
665
X00779


 



O. volvulusa
GAtatGaGAC AAACaGTTGc GGT
−2359
M64333


 



Humana
GAtatGaGAC AGACaGTTGc TGT
−2360
X03558


 



G. max B1b
GAAGGAGGCA AAACTGTTGG AGC
−2361
Y15107


 



G. max B2b
GAAGGAGGCA AAACTGTTGG AGC
−2362
Y15108


 



E. colic
GAAGGCGGCC GTACcGTTGG CGC
78
—


 



S. aureofaciensc
GAGGGTGGTC GTACcGTgGG CGC
−2363
AF007125


 



E. tenellab
GAAGGAGGAA AAACTATAGG AGC
−2364
AI755521


 



T. gondiib
GAAGGAGGTC GTACTATAGG AGC
−2365
Y11431


 



S. cerevisiaeb
GAGGGTGGAA GAACTGTTGG TAC
619
K00428


 



A. thalianab
GAAGGAGGTA GAACaGTTGG AGC
−2366
X89227


 



Selected sequence






for universal






primer





 



Selected sequences
GARGGIGGIM RIACTRTWGGd
652




for universal
GAGGGGYGGCC GITCIGTd
561




primers





The sequence numbering refers to the Saccharomyces cerevisiae tuf (M) gene (SEQ ID NO. 619).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches for SEQ ID NOs. 652 and 664 are indicated by lowercase letters.


Mismatches for SEQ ID NO. 561 are indicated by underlined nucleotides.


Dots indicate gaps in the sequences displayed.


“R” “Y” “M” “K” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“M” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.


aThis sequence refers to tuf(EF-1) gene.


bThis sequence refers to tuf (M) or organelle gene.


cThis sequence refers to tuf gene from bacteria.


dThese sequences are the reverse-complement of the selected primers.













TABLE 45







Strategy for the selection of eukaryotic sequencing primers from tuf (EF-1) sequences.












SEQ ID
Accession



154                       179   286                          314
NO.:
#:





S. cerevisiae
GG TTCTTTCAAG TACGCTTGGG TTTT...AGAGA TTTCATCAAG AACATGATTA CTGG...
665
X00779


 


B. hominis
GG CTCCTTCAAG TACGCGTGGG TGCT...CGTGA CTTCATaAAG AACATCATCA CTGG...
−2367
D64080


 


C. albicans
GG TTCTTTCAAA TACGCTTGGG TCTT...AGAGA TTTCATCAAG AATATGATCA CTGG...
−2368
M29934


 


C. neoformans
TC TTCTTTCAAG TACGCTTGGG TTCT...CGAGA CTTCATCAAG AACATGATCA CCGG...
−2369
U81803


 


E. histolytica
GG ATCATTCAAA TATGCTTGGG TCTT...AGAGA TTTCATTAAG AACATGATTA CTGG...
−2370
M92073


 


G. lamblia
GG CTCCTTCAAG TACGCGTGGG TCCT...CGCGA CTTCATCAAG AACATGATCA CGGG...
−2371
D14342


 


H. capsulatum
AA ATCCTTCAAA TATGCGTGGG TCCT...CGTGA CTTCATCAAG AACATGATCA CTGG...
−2372
U14100


 


Human
GG CTCCTTCAAG TATGCCTGGG TCTT...AGAGA CTTtATCAAA AACATGATTA CAGG...
−2373
X03558


 


L. braziliensis
GC GTCCTTCAAG TACGCGTGGG TGCT...CGCGA CTTCATCAAG AACATGATCA CCGG...
−2374
U72244


 


O. volvulus
GG CTCATTTAAA TATGCTTGGG TATT...CGTGA TTTCATTAAG AATATGATCA CAGG...
−2375
M64333


 


P. berghei
GG TagTTTCAAA TATGCATGGG TTTT...AAAcA TTTtATTAAA AATATGATTA CTGG...
−2376
AJ224150


 


P. knowlesi
GG AagTTTTAAG TACGCATGGG TGTT...AAGGA TTTCATTAAA AACATGATTA CCGG...
−2377
AJ224153


 


S. pombe
GG TTCCTTCAAG TACGCCTGGG TTTT...CGTGA TTTCATCAAG AACATGATTA CCGG...
−2378
U42189


 


T. cruzi
TC TTCTTTCAAG TACGCGTGGG TCTT...CGCGA CTTCATCAAG AACATGATCA CGGG...
−2379
L76077


 


Y. lipolytica
GG TTCTTTCAAG TACGCTTGGG TTCT...CGAGA TTTCATCAAG AACATGATCA CCGG...
−2380
AF054510


 


Selected sequences
    TCITTYAAR TAYGCITGGG T
558



for amplification
                                   GA YTTCATYAAR AAYATGATYA C
560



primers
                                    GA YTTCATIAAR AAYATGAT
653








The sequence numbering refers to the Saccharomyces cerevisiae tuf (EF1) gene fragment (SEQ ID NO. 665).


Nucleotides in capitals are identical to the selected sequences SEQ ID NOs. 558, 560 or 653,


or match those sequences.


Mismatches for SEQ ID no. 558 and 560 are indicated by lowercase letters.


Mismatches for SEQ ID NO. 653 are indicated by underlined nucleotides.


Dots indicate gaps in the sequences displayed.


“R” “Y” “M” “K” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“M” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides


A, C, G or T.















SEQ ID
Accession



   751                     776    1276                        1304
NO.:
#:





S. cerevisiae
...GTTTACAA GATCGGTGGT ATTGGTAC...GACATG AGACAAACTG TCGCTGTCGG TGT
665
X00779


 


B. hominis
...GTGTACAA GATTGGCGGT ATTGGTAC...GATATG AGACAGACTG TCGCTGTCGG TAT
−2381
D64 080


 


C. albicans
...GTTTACAA GATCGGTGGT ATTGGTAC...GATATG AGACAAACCG TTGCTGTtGG TGT
−2382
M29934


 


C. neoformans
...GTCTACAA GATCGGTGGT ATCGGCAC...GACATG CGACAGACCG TTGCCGTtGG TGT
−2383
U81803


 


E. histolytica
...GTTTACAA GATTTCAGGT ATTGGAAC...GATATG AaACAAACCG TTGCTGTtGG AGT
−2384
M92073


 


G. lamblia
...GTCTACAA GATCTcGGGc gTCGGGAC...~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~
−2385
D14342


 


H. capsulatum
...GTGTACAA AATCTcTGGT ATTGGCAC...GACATG AGACAAACCG TCGCTGTCGG TGT
−2386
U14100


 


Human
...GTCTACAA AATTGGTGGT ATTGGTAC...GATATG AGACAGACAG TTGCgGTgGG TGT
−2387
X03558


 


L. braziliensis
...GTGTACAA GATCGGCGGT ATCGGCAC...GACATG CGCagAACGG TCGCCGTCGG CAT
−2388
U72244


 


O. volvulus
...GTTTACAA AATTGGAGGT ATTGGAAC...GATATG AGACAAACAG TTGCTGTtGG CGT
−2389
M64333


 


P. berghei
...GTATACAA AATTGGTGGT ATTGGTAC...GATATG AGACAAACAA TTGCTGTCGG TAT
−2390
AJ224150


 


P. knowlesi
...GTATACAA AATCGGTGGT ATTGGTAC...GATATG AGACAAACCA TTGCTGTCGG TAT
−2391
AJ224153


 


S. pombe
...GTTTACAA GATCGGTGGT ATTGGTAC...GACATG CGTCAAACCG TCGCTGTCGG TGT
−2392
U42189


 


T. cruzi
...GTGTACAA GATCGGCGGT ATCGGCAC...GACATG CGCCAGACGG TCGCCGTCGG CAT
−2393
L76077


 


Y. lipolytica
...GTCTACAA GATCGGTGGT ATCGGCAC...GACATG CGACAGACCG TTGCTGTCGG TGT
−2394
AF054510


 


Selected sequence
      TACAA RATYKGIGGT ATYGG
654



for amplification





primer





 


Selected sequences
      TACAA RATYKGIGGT ATYGG
655



for amplification
                                     ATG MGICARACIR TYGCYGTCGG
559



primersa





The sequence numbering refers to the Saccharomyces cerevisiae tuf (EF-1) gene fragment (SEQ ID NO. 665).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


“~” indicate incomplete sequence data.


Dots indicate gaps in the sequences displayed.


“R” “Y” “N” “K” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“N” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.


aThis sequences are the reverse-complement of the selected primers.













TABLE 46







Strategy for the selection of Streptococcus agalactiae-specific


amplification primers from tuf sequences.












SEQ ID
Accession



305                           334   517                       542
NO.:
#:













S. agalactiae
CCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG
207
—


 


S. agalactiae
CCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG
208
—


 


S. agalactiae
CCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG
209
—


 


S. agalactiae
CCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG
210
—


 


S. anginosus
CCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AACGTaGGgG TTCTTCTTCG TG
211
—


 


S. anginosus
CCAGAA CGTGATACTG ACAAACCaTT gCTT...AGAt AACGTaGGgG TTCTTCTTCG TG
221
—


 


S. bovis
CCAaAA CGTGATACTG ACAAACCaTT gCTT...GGAt AACGTTGCTG TTCTTCTTCG TG
212
—


 


S. gordonii
CCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AAtGTaGGTG TcCTTCTTCG TG
223
—


 


S. mutans
CCAGAA CGTGATACTG ACAAgCCgcT cCTT...GGAt AAtGTTGGTG TTCTcCTTCG TG
224
—


 


S. pneumoniae
CCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AACGTaGGTG TcCTTCTTCG TG
145a
—


 


S. sanguinis
CCAGAA CGcGATACTG ACAAgCCaTT gCTT...GGAC AACGTaCGTG TgCTTCTcCG TG
227
—


 


S. sobrinus
CCAaAA CCcGATACTG AtAAgCCaTT gCTT...AGAt AACGTTGGTG TgCTTCTTCG TG
228
—


 


B. cepacia
CCGGAg CGTGcagtTG ACggcgCgTT cCTG...CGAC AACGTTGGTa TcCTgCTgCG cG
16
—


 


B. fragilis
CCTccg CGcCATgtTG AtAAACCTTT ctTG...TGAC AACGTaGGTc TgtTgcTTCG TG
−2395 
P33165


 


B. subtilis
CCAGAA CGcGAcACTG AaAAACCaTT caTG...TGAC AACaTTGGTG ccCTTCTTCG cG
−2396 
Z99104


 


C. diphtheriae
CCAGAg CGTGAgACcG ACAAgCCaTT cCTC...CGAC AACtgTGGTc TgcTTCTcCG TG
662
—


 


C. trachomatis
CCAGAA aGaGAaAtTG ACAAgCCTTT cTTA...AGAg AAtGTTGGat TgcTcCTcaG aG
22
—


 


E. coli
CcAGAg CGTGcgAtTG ACAAgCCgTT cCTg...TGAg AACGTaGGTG TTCTgCTgCG TG
78
—


 


G. vaginalis
CCAact CacGATctTG ACAAgCCaTT cTTg...CGAt RACacTGGTc TTCTTCTcCG cG
135a
—


 


S. aureus
CCAGAA CGTGATtCTG ACAAACCaTT cATg...TGAC AACaTTGGTG catTatTaCG TG
179
—


 


Selected sequence
   GAA CGTGATACTG ACAAACCTTT A
549



for species-





specific primer





 


Selected sequence
                                       C AACGTTGGTG TTCTTCTTC
550



for species-





specific primerb





The sequence numbering refers to the Streptococcus agalactiae tuf gene fragment (SEQ ID NO. 209).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequences displayed.


“R” “Y” “M” “K” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“M” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.


aSEQ ID NO. refers to previous patent publication WO98/20157.


bThis sequence is the reverse-complement of the selected primer.













TABLE 47







Strategy for the selection of Streptococcus agalactiae-specific


hybridization probes from tuf sequences.











401                            431 433
SEQ ID NO.:
Accession #:





S. acidominimus
GGTACTGT TaaaGTtAAt GACGAAGTTG AAATCGTTGG TATcAAAGAc
206
—


 


S. agalactiae
GGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA
209
—


 


S. agalactiae
GGTACTGT TCGTGTCAAC CACGAAGTTG AAATCGTTGG TATTAAAGAA
2397
—


 


S. agalactiae
GGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA
207
—


 


S. agalactiae
GGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA
210
—


 


S. agalactiae
GGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA
208
—


 


S. anginosus
GGTACTGT TaaaGTCAAC GACGAAGTTG AAATCGTTGG TATccgtGAt
211
—


 


S. anginosus
GGTACTGT TaaaGTCGAC GAtGAAGTTG AAATCGTTGG TATCcgcGAg
221
—


 


S. bovis
GGTACTGT TaaaGTCAAC GACGAAGTTG AAATCGTTGG TATccgtGAc
212
—


 


S. anginosus
GGTACTGT TaaaGTCAAt GAtGAAGTTG AAATtGTTGG TATTCgtGAc
213
—


 


S. cricetus
GGTACTGT TaagGTCAAt GACGAAGTTG AAATCGTTGG TATcAAgGAc
214
—


 


S. cristatus
GGTACTGT TCGTGTCAAC GAtGAAaTcG AAATCGTTGG TATCAAAGAA
215
—


 


S. downei
GGTACTGT TaagGTCAAC GACGAAGTTG AAATCGTTGG TATcAAgGAC
216
—


 


S. dysgalactiae
GGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATCAAAGAA
217
—


 


S. equi equi
GGTACTGT TCGTGTtAAC GACGAASTcG AAATCGTTGG TATcAgAGAc
218
—


 


S. ferus
GGTACTGT aaGaGTCAAC GAtGAAGTTG AAATCGTTGG TATcAAAGAC
219
—


 


S. gordonii
GGTAtcGT TaaaGTCAAt GACGAAaTcG AAATCGTTGG TATcAAAGAA
220
—


 


S. macacae
GGTACTGT TaagGTtAAt GAtGAAGTTG AAATCGTTGG TATTcgtGAc
222
—


 


S. gordonhi
GGTAtcGT TaaaGTCAAC GACGAAaTCG AAATCGTTGG TATCAAAGAA
223
—


 


S. mutans
GGTACTGT TaaaGTtAAC GAtGAAGTTG AAATCGTTGG TATccgtGAt
224
—


 


S. oralis
GGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATCAAAGAA
−2398
233170


 


S. parasanguinis
GGTgtTGT TCGTGTCAAt GAtGAAaTcG AAATCGTTGG TATcAAAGAA
225
—


 


S. pneumnoniae
GGTAtcGT TaaaGTCAAC GACGAAaTCG AAATCGTTGG TATcAAAGAA
2399
—


 


S. pyogenes
GGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA
−2400
Genome project


 


S. ratti
GGTACTGT TaaaGTCAAt GACGAAGTTG AAATCGTTGG TATccgtGAt
226
—


 


S. salivarius
GGTgtTGT TCGTGTCAAt GACGAAGTTG AAATCGTTGG TCTTAAAGAA
2401
—


 


S. sanguinis
GGTAtcGT TaaaGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA
227
—


 


S. sobrinus
GGTACTGT TaagGTtAAC GACGAAGTTG AAATCGTTGG TATccgtGAc
228
—


 


S. suis
GGTACTGT TCGTGTCAAC GACCAAaTcG AAATCGTTGG TcTTcAAGAA
229
—


 


S. uberis
GGTACTGT TCGTGTCAAC GACGAAaTTG AAATCGTTGG TATcAAAGAA
230
—


 


S. vestibularis
GGTgtTGT TCGTGTtAAt GACGAAGTTG AAATCGTTGG TcTTAAAGAA
231
—


 


Selected sequences
   ACTGT TCGTGTCAAC GACGAAGTTG AAA
582



for species-specific
                                   CGTTGG TATTAAAGAA
583



hybridiation probesb






                     470
SEQ ID NO.:
Accession #:





S. acidominimus
GAaATCtctA AAGCGGTTGT TA
206
—


 


S. agalactiae
GATATCCAAA AAGCAGTTGT TA
209
—


 


S. agalactiae
GATATCCAAA AAGCAGTTGT TA
2397
—


 


S. agalactiae
GATATCCAAA AAGCAGTTGT TA
207
—


 


S. agalactiae
GATATCCAAA AAGCAGTTGT TA
210
—


 


S. agalactiae
GATATCCAAA AAGCAGTTGT TA
208
—


 


S. anginosus
GAaATCCAAA AAGCAGTTGT TA
211
—


 


S. anginosus
GAaATCCAAA AAGCAGTTGT TA
221
—


 


S. bovis
GAcATCCAAA AAGCtGTTGT TA
212
—


 


S. anginosus
GAAATCCAAA AAGCAGTTGT TA
213
—


 


S. cricetus
GAeATCCAAA AAGCgGTTGT TA
214
—


 


S. cristatus
GAaATCCAAA AAGCAGTTGT TA
215
—


 


S. downei
GAaATCCAAA AAGCAGTTGT TA
216
—


 


S. dysgalactiae
GAaActaAAA AAGCtGTTGT TA
217
—


 


S. equi equi
GAgATCaAAA AAGCAGTTGT TA
218
—


 


S. ferus
GAaATCactA AAGCAGTTQT TA
219
—


 


S. gordonii
GAaATCCAAA AAGCAGTTGT TA
220
—


 


S. macacae
GATATtCAAA AAGCAGTTGT TA
222
—


 


S. gordonhi
GAaActCAAA AAGCAGTTGT TA
223
—


 


S. mutans
GAcATtCAAA AAGCtGTTGT TA
224
—


 


S. oralis
GAaActCAAA AAGCAGTTGT TA
−2398
233170


 


S. parasanguinis
GAaATCCAAA AAGCAGTTGT TA
225
—


 


S. pneumnoniae
GAaActCAAA AAGCAGTTGT TA
2399
—


 


S. pyogenes
GAaActaAAA AAGCtGTTGT TA
−2400
Genome project


 


S. ratti
GAcATCCAAA AAGCtGTTGT TA
226
—


 


S. salivarius
GACATCCAAA AAGCAGTTGT TA
2401
—


 


S. sanguinis
GAaATCCAAA AAGCAGTTGT TA
227
—


 


S. sobrinus
GATATCCAAA AAGCAGTTGT TA
228
—


 


S. suis
GAaAaatctA AAGCAGTTGT TA
229
—


 


S. uberis
GAaActaAAA AAGCAGTTGT TA
230
—


 


S. vestibularis
GAaATCCAAA AAGCAGTTGT TA
231
—


 


Selected sequences





for species-specific
GATATCCAAA AAGCAGTTG
583



hybridization probesb





The sequence numbering refers to the Streptococcus agalactiae tuf gene fragment (SEQ ID NO. 209).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequences displayed.


bThese sequences are the reverse-complement of the selected probes.













TABLE 48







Strategy for the selection of Streptococcus agalactiae-specific


amplification primers from atpD sequences.










39                                          80   203
SEQ ID NO.:





S. agalactiae
TT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG
380


 


S. agaiactlae
TT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG
379


 


S. agaiactlae
TT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG
381


 


S. agaiactlae
TT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG
382


 


S. agaiactlae
TT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG
383


 


S. bovis
TT GATTGTtTAT AAAAATGGCG ATAAGTCtCA AAAAaTAGTg...TAAaGAaA CTTTGGGTCG
−2402a


 


S. salivarius
TT GgTCGTtTAT ActgATGaac AaAAGTCtaA AcgtaTcGTg...TAAaGATA CccTtGGaCG
387


 


S. pneumoniae
cT tgTCGTCTAC AAAgATGaCG AaAgaaaAac AAAAaTcGTc...TAAaGAaA CTTTGGGaCG
−2403b


 


S. pyogenes
TT GATTGTtTAT AAAgATaGtG ATAAaaagCA AAAAaTcGTc...TAAaGAaA CTTTGGGaCG
−2404c 


 


S. anginosus
cT tgTaGTCTAT AAAAATGaCG AaAktaaAtc AAAAaTcGTc...gAAaGAaA CacTtGGTCG
386


 


S. sanguinis
cT tgTaGTCTAT AAAAATGatG AgAAaaaAtc AAAAaTcGTc...aAAGGAaA CTcTaGGcCG
−2405d


 


S. mutans
TT GgTcGTtTAT AAAgATGGCG AcAAGTCtCA AAgAaTtGTt...aAAaGAaA CacTaGGTCG
−2406e 


 


B. anthracis
gT aAaacagagc AAcgAaaaCG gaAcaagcat tAActTkacA...TgAtGcaA CacTtGGTCG
247


 


B. cereus
gT akaacaaagc AAcgAaaaCG g...aagcat gAActTAacA...TgAtGcaA CacTtGGaCG
248


 


E. faecium
TT agTTGTtTAT AAAAATGaCG AaAAtaaAtc AAAAGTtGTt...TAAaGAaA CaTTaGGTCG
292


 


E. galilnarum
TT GATcGTtTAc AAAAAaGaCG AgAAaaaAac AAAAGTAGTA...aAcaGATA CTCTaGGcCG
293


 


S. faecalis
TT agTcGTtTAT AAAAATGGCG AagcaaaACA AAAAGTAGTA...TAAaGATA CaTTaGGTCG
291


 


E. coli
Ta cgaTGctctT gAggtgcaaa ATggtaatgA gcgtcTgGTg...TAAaGcgA CTcTGGGcCG
−f2407


 


L. monocytogenes
Ta tAaatctgAT gcAgAaGaaG cacccaCtag ccAAcTtact...TAcaGtaA CTcTtGGTCG
324


 


S. aureus
gT tATTGatgtg cctAAaGaaG AaggtaCAat AcAAcTAacA...TgAtGAaA CaTTaGGTCG
366


 


S. epidermidis
ca cATcGaagtT cctAAaGaaG ATggagcgCt tcAAtTAacA...TgAcGtaA CTcTaGGaaG
370


 


Selected sequences
    ATTGTCTAT AAAAATGGCG ATAAGTC
627


or species-
              AAAATGGCG ATAAGTCACA AAAAGTA
628


specific primer




 


Selected sequences
                                                    GGATA CTTTGGGTCG
625


for species-




specific primersg















            234   368                             399
SEQ ID NO.:






S. agalactiae
TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG
380


 



S. agaiactlae
TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG
379


 



S. agaiactlae
TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG
381


 



S. agaiactlae
TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG
382


 



S. agaiactlae
TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG
383


 



S. bovis
TGTgTTtAAt GTTC...CcT tcTtGCcCCT TACcTAAAAG GTGGTAAAG
−2402a


 



S. salivarius
TGTCTTtAAC GTTC...CTT gcTAGCcCCT TACcTtAAgG GTGGTAAAG
387


 



S. pneumoniae
TGTCTTCAAC GTTt...CcT tcTtGCcCCT TACcTtAAAG GTGGThAAG
−2403b


 



S. pyogenes
cGTCTTtAAt GTaC...CcT tcTtGCCCCT TACcTtAAAG GTGGTAAAG
−2404c 


 



S. anginosus
cGTCTTtAAC GTTt...CcT tTTAGCcCCc TACcTcAAAG GTGGgAAAG
386


 



S. sanguinis
gGTgTTCAAt GTTt...CcT gcTAGCACCT TAtcTgAAAG GTGGgAAAG
−2405d


 



S. mutans
TGTCTTtAAt GTTC...CcT tcTtGCcCCT TAtcTtAAAG GTGGTAAAG
−2406e 


 



B. anthracis
TGTaTTtAAC GTaT...CTT AcTtGCtCCT TACaTtAAgG GTGGTAAga
247


 



B. cereus
TGTaTTCAAC GTat...CTT AcTtGCtCCT TACaTtAAgG GTGGTAAga
248


 



E. faecium
cGTaTTCAAC GTaC...tTT gcTtGCcCCa TAtTTAAAAG GTGGgAAAG
292


 



E. galilnarum
aGTaTTtAAt GTaC...tTT ATTAGCtCCT TACTTAAAAG GTGGTAAAG
293


 



S. faecalis
TGTgTTtAAC GTTt...CTT ATTAGCACCT TAtcTAAAAG GTGGTAAAG
291


 



E. coli
TaTCaTgAAc GTaC...CcT gaTgtgtCCg TtCgctAAgG GcGGTAAAG
−f2407


 



L. monocytogenes
TGTaTTtAAt GTat...CTT gcTAGCtCCT TACTTAAAAG GTGGTAAAa
324


 



S. aureus
TGTaTTtAAt GTaC...tTT AcTAGCACCT TAtaTtAAAG GTGGTAAAa
366


 



S. epidermidis
aGTgTTtAAC GTaC...CTT ATTAGCACCT TACaTAAAAG GTGGTAAAa
370


 



Selected sequences





or species-





specific primer




 



Selected sequences
TGTCTTCAAC G
625



for species-
                      ATTAGCACCT TACTTAAAAG GTGGTA
626



specific primersg





The sequence numbering refers to the Streptococcus agallactiae tuf gene fragment (SEQ ID NO. 380).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lower-case letters.


Dots indicate gaps in the sequences displayed.


a, d, e, f These sequences were obtained from Genbank and have accession #: a = AB009314, d = AF001955, e = U31170, and f = V00311.


b, c These seouences were obtained from qenome sequencing projects,


g These secquences are the reverse-complement or the selected primers.













TABLE 49







Strategy for the selection of Candida albicans/dabliniensis-specific


amplification primers, Candida albicans-specific hybridization probe


and Candida dubliniensis-specific hybridization probe from tuf sequences.












SEQ ID




337                             368   403
NO.
Accession #:





C. albicans
CGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC
624
—


 


C. albicans
CGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC
409
—


 


C. albicans
CGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC
410
—


 


C. albicans
CGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC
407
—


 


C. albicans
CGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC
408
—


 


C. dubliniensis
CGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC
412
—


 


C. dubliniensis
CGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTc
414
—


 


C. dubliniensis
CGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC
415
—


 


C. glabrata
CATC AAGAAGGTcG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCcaC
417
—


 


C. guilliermondii
CGTC AAGAAGGTTG GTTACAACCC tAAGACTG...CAACATGA TTGAggCtTC
418
—


 


C. kefyr
CATC AAGAAGGTcG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCcaC
421
—


 


C. krusei
CATC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCATC
422
—


 


C. lusitaniae
CGTC AAGAAGGTTG GTTACAACCC tAAGACTG...CAACATGA TTGAgCCATc
424
—


 


C. neoformans
CATC AAGAAGGTTG GTTACAACCC CAAGgCTG...CAACATGt TgGAggagaC
623
—


 


C. parapsilosis
CGTC AAGAAGGTTG GTTACAACCC tAAagCTG...CAAtATGA TTGAACCATC
426
—


 


C. tropicalis
CGTC AAGAAGGTTG GTThCAACCC tAACgCTG...CAACATGA TTGAAgCtTC
429
—


 


A. fumigatus
CATC AAGAAGGTCG GCTACAACCC cAAGgCCG...CAACATGc TTGAgCCcTC
404
—


 


Human
CATt AAGAAaaTTG GcTACAACCC CgAcACAG...CAACATGc TgGAgCCAag
−2408
X03558


 


P. anomale
TATC AAGAAaGTTG GTTACAACCC AAAaACTG...TAACATGA TTGAACCATC
447
—


 


S. cerevisiae
TATC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtaC
622
—


 


S. pombe
CATC AAGAAGGTcG GTTtCAACCC cAAGACCG...TAACATGA TTGAgCCcAC
−2409
U42189


 


Selected sequence
   C AAGAAGGTTG GTTACAACCC AAAGA




for species-specific





amplification primera





 


Selected sequence





for species-specific





amplification primera, b





 


Selected sequences
                                         CATGA TTGAACCtTC
577



for species-specific
                                         CATGA TTGAAGCTTC
578



hybridization probes

















SEQ ID





     428   460                              491
NO.:
Accession #:






C. albicans
CACCAACT...C AAATCCGGTA AAGTTACTCG TAAGACCTTG T
624
—


 



C. albicans
CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T
409
—


 



C. albicans
CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T
410
—


 



C. albicans
CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T
407
—


 



C. albicans
CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T
408
—


 



C. dubliniensis
CACCAACT...C AAATCCGGTA AgGTTACTGG TAAGACCTTG T
412
—


 



C. dubliniensis
CACCAACT...C AAATCCGGTA AgGTTACTGG TAAGACCTTG T
414
—


 



C. dubliniensis
CACCAACT...C AAATCCGGTA AgGTTACTGG TAAGACCTTG T
415
—


 



C. glabrata
CACCAACG...C AAggCtGGTg tcGTcAagGG TAAGACCTTG T
417
—


 



C. guilliermondii
tACCAACT...C AAggCtGGTA AgtccACcGG TAAGACtTTG T
418
—


 



C. kefyr
CACCAACG...C AAggCtGGTA ccGTcAagGG TAAGACCTTG T
421
—


 



C. krusei
CACCAACT...C AAggcaGGTg ttGTTAagGG TAAGACCTTA T
422
—


 



C. lusitaniae
YACCAACT...C AAgTCYGGTA AgtccACcGG TAAGACCTTG T
424
—


 



C. neoformans
CACCAAGT...C AAgTCtGGTg tttccAagGG TAAGACCcTC C
623
—


 



C. parapsilosis
aACCAACT...T AAAgCtGGTA AgGTTACcGG TAAGACCTTG T
426
—


 



C. tropicalis
tACCAACT...C AAggCtGGTA AqGTTACcGG TAAGACtTTG T
429
—


 



A. fumigatus
CtCCAACT...C AAggCCGGcA AgGTcACTGG TAAGACCcTC A
404
—


 



Human
tgCtAACA...T AAggatGGcA AtGcCAgTCG aAccACgcTG C
−2408
X03558


 



P. anomale
aWCtAACT...C AAAgctGGTg AAGcTAaacG TAAaACtTTA T
447
—


 



S. cerevisiae
CACCAACG...C AAggcCGGtg tcGTcAagGG TAAGACtTTG T
622
—


 



S. pombe
CACCAACA...C AAggCtGGTg tcGTcAagGG TAAGACtcTT T
−2409
U42189


 



Selected sequence






for species-specific






amplification primera





 



Selected sequence
               ATCCGGTA AAGTTACTGG TAAGACCT





for species-specific






amplification primera, b





 



Selected sequences
CACCA (C. albicans)
577




for species-specific
CACCA (C. dublinensis)
578




hybridiaation probes





The sequence nunbering refers to the Candida albicans tuf gene fragment (SEQ ID NO. 408).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches for SEQ ID NO. 577 are indicated by lower-case letters.


Mismatches for SEQ ID NO. 578 are indicated by underlined nucleotides


Dots indicate gaps in the sequences displayed.


“R” “Y” “M” “K” “W” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“M” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.


aC. albicans primers have been described in a previous patent (publication WO98/20157, SEQ ID NOs. 11-12)


bThis sequence is the reverse-complement of the selected primer.













TABLE 50







Strategy for the selection of Staphylococcus-specific


amplification primers from tuf sequences.











310                            340   652
SEQ ID NO.
Accession #: 





S. aureus
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTACTG
179
—


 


S. aureus
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACC~ ~~~~~~~~~~
176
—


 


S. aureus
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTMCTG
177
—


 


S. aureus aureus
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTACTG
180
—


 


S. auricularis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...ActTTACCA GAAGGTACaG
181
—


 


S. capitis capitis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA CAAGGTACTG
182
—


 


M. caseolyticus
A CTGGaCGTGT TGAgCGTGGa CAAATCAAAG...AACTTACCA GAAGGTACTG
183
—


 


S. cohnii
A CAGGgCGTGT TGAACCTGGT CAAATCAAAG...ActTTACCA GAAGGTACTG
184
—


 


S. epidermidis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...~~~~~~~~~ ~~~~~~~~~~
185
—


 


S. epidermidis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACaG
141a
—


 


S. haemoiyticus
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTC
186
—


 


S. haemoiyticus
A CAGGtCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAG~~~~~~
188
—


 


S. haemolytlcus
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG
189
—


 


S. hominis hominis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG
191
—


 


S. hominis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG
193
—


 


S. hominis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGG~~~~~
194
—


 


S. hominis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG
195
—


 


S. hominis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG
196
—


 


S. lugdunensis
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACaG
197
—


 


S. saprophyticus
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...~~~~~~~~~ ~~~~~~~~~~
198
—


 


S. saprophyticus
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG
199
—


 


S. saprophyticus
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG
200
—


 


S. sciuri sciuri
A CAGGCCGTGT TGAACGTGGT CAAATCACTG...AACTTACCA GAAGGTACTG
201
—


 


S. warneri
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CAaTTACCA GAAGGTACTG
187
—


 


S. warneri
A CAGGCCGTGT TGAACCTGGT CAAATCAAAG...~~~~~~~~~ ~~~~~~~~~~
192
—


 


S. warneri
A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CAaTTACCA GAAGGTACTG
202
—


 


B. subtills
A CTGGCCGTGT aGAACGcCGa CAAATCAAAG...CAtCTtCCA GAAGGcgtaG
−2410
Z99104


 


E. coli
A CCGGtCGTGT aGAACGcGGT atcATCAAAG...GAaCTgCCg GAAGGcgtaG
78
—


 


L. monocytogenes
A CTGGaCGTGT TGAACGTGGa CAAgTtAAAG...AcacTtCCA GAAGGTACTG
2411
—


 


Selected sequence
    GGCCGTGT TGAACCTCGT CAAATCA
553



for genus-specific





primer





 


Selected sequences
                                        TTACCA GAAGGTACTG
575



for genus-specific
                                        TTACCA GAAGGTACTG
707



primersb
















          682
SEQ ID NO.:
Accession #:






S. aureus
AAATGGTAAT GC
179
—


 



S. aureus
~~~~~~~~~~ GC
176
—


 



S. aureus
AAATGGTAAT GC
177
—


 



S. aureus aureus
AAATGGTAAT GC
180
—


 



S. auricularis
AAATGGTAAT GC
181
—


 



S. capitis capitis
AAATGGTTAT GC
182
—


 



M. caseolyticus
AAATGGTAAT CC
183
—


 



S. cohnii
AAATGGTTAT GC
184
—


 



S. epidermidis
~~~~~~~~~~ ~~
185
—


 



S. epidermidis
AAATGGTTAT GC
141a
—


 



S. haemoiyticus
AAATGGTTAT GC
186
—


 



S. haemoiyticus
~~~~~~~~~~ ~~
188
—


 



S. haemolytlcus
AAATGG~~~~ ~~
189
—


 



S. hominis hominis
AAATGGTAAT GC
191
—


 



S. hominis
AAATGGTAAT CC
193
—


 



S. hominis
~~~~~~~~~~ ~~
194
—


 



S. hominis
AAATGGTAAT CC
195
—


 



S. hominis
AAATGGTAAT GC
196
—


 



S. lugdunensis
AAATGGTTAT GC
197
—


 



S. saprophyticus
~~~~~~~~~~ ~~
198
—


 



S. saprophyticus
AAATGGTTAT GC
199
—


 



S. saprophyticus
AAATGGTTAT GC
200
—


 



S. sciuri sciuri
AAATGGTTAT GC
201
—


 



S. warneri
~~~~~~~~~~ ~~
187
—


 



S. warneri
~~~~~~~~~~ ~~
192
—


 



S. warneri
AAATGGTTAT CC
202
—


 



B. subtills
AAATGGTTAT GC
−2410
Z99104


 



E. coli
AgATGGTAAT CC
78
—


 



L. monocytogenes
AAATGGTAAY GC
2411
—


 



Selected sequence






for genus-specific






primer





 



Selected sequences
AAATGGTIA
575




for genus-specific
AAATGGTWA
707




primersb





The sequence numbering refers to the Staphylococcus aureus tuf gene fragment (SEQ ID NO. 179).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


“~” indicate incomplete sequence data.


Dots indicate gaps in the sequences displayed.


“R” “Y” “N” “K” “W” and “S” designate mucleotide positions which are degenerated.


“R” stands for A or C;


“Y” stands for C or T;


“M” stands for A or C;


“K” stands for G or T;


“N” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.


bThese sequences are the reverse-complement of the selected primers.













TABLE 51







Strategy for the selection of the Staphylococcus-


specific hybridization probe from tuf sequences.











400                       425
SEQ ID NO.:
Accession #:





S. aureus
G TTGAAATGTT CCGTAAATTA TTAGA
179
—


 


S. aureus
G TTGAAATGTT CCGTAAATTA TTAGA
176
—


 


S. aureus
G TTGAAATGTT CCGTAAATTA TTAGA
177
—


 


S. aureus
G TTGAAATGTT CCGTAAATTA TTAGA
178
—


 


S. aureus aureus
G TTGAAATGTT CCGTAAATTA TTAGA
180
—


 


S. auricularis
G TAGAAATGTT CCGTAAATTA TTAGA
181
—


 


S. capitis capitis
G TAGAAATGTT CCGTAAATTA TTAGA
182
—


 


M. caseolyticus
G TAGAAATGTT CCGTAAATTA TTAGA
183
—


 


S. cohnii
G TAGAAATGTT CCGTAAATTA TTAGA
184
—


 


S. epidermidis
G TAGAAATGTT CCGTAAATTA TTAGA
185
—


 


S. haemolyticus
G TAGAAATGTT CCGTAAATTA TTAGA
186
—


 


S. haemolyticus
G TAGAAATGTT CCGTAAATTA TTAGA
189
—


 


S. haemolyticus
G TAGAAATGTT CCGTAAATTA TTAGA
190
—


 


S. haemolyticus
G TAGAAATGTT CCGTAAATTA TTAGA
188
—


 


S. hominis
G TAGAAPTGTT CCGTAAATTA TTAGA
196
—


 


S. hominis
G TAGAAATGTT CCGTAAATTA TTAGA
194
—


 


S. hominis hominis
G TAGAAATGTT CCGTAAATTA TTAGA
191
—


 


S. hominis
G TAGAAATGTT CCGTAAATTA TTAGA
193
—


 


S. hominis
G TAGAAATGTT CCGTAAATTA TTAGA
195
—


 


S. lugdunensis
G TAGAAATGTT CCGTAAATTA TTAGA
197
—


 


S. saprophyticus
G TAGAAATGTT CCGTAAATTA TTAGA
198
—


 


S. saprophyticus
G TAGAAATGTT CCGTAAATTA TTAGA
200
—


 


S. saprophyticus
G TAGAAATGTT CCGTAAATTA TTAGA
199
—


 


S. sciuri sciuri
G TTGAAATGTT CCGTAAATTA TTAGA
201
—


 


S. warneri
G TAGAAATGTT CCGTAAgTTA TTAGA
187
—


 


S. warneri
G TAGAAATGTT CCGTAAgTTA TTAGA
192
—


 


S. warneri
G TAGAAATGTT CCGTAAgTTA TTAGA
202
—


 


S. warneri
G TAGAAATGTT CCGTAAgTTA TTAGA
203
—


 


B. subtilis
G TTGAAATGTT CCGTAAgcTt cTTGA
−2412
Z99104


 


E. coli
G TTGAAATGTT CCGcAAAcTg cTGGA
78
—


 


L. monocytogenes
G TAGAAATGTT CCGTAAATTA cTAGA
2413
—


 


Selected sequence
    GAAATGTT CCGTAAATTA TT
605



for genus-specific





hybridization probe





The sequence numbering refers to the Staphylococcus aureus tuf gene fragment (SEQ ID NO. 179).


Nucleotides in capitals are identical to the selected sequence or match that sequence.


Mismatches are indicated by lowercase letters.













TABLE 52







Strategy for the selection of Staphylococcus saprophyticus-


specific and of Staphylococcus haemolyticus-specific


hybridization probes from tuf sequences.










339                                            383
SEQ ID NO.:





S. aureus
AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA
179


 


S. aureus
AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA
176


 


S. aureus
AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA
177


 


S. aureus
AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA
178


 


S. aureus aureus
AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGkCACaTC TAA
180


 


S. auricularis
AG TCGGTGAAGA AgTtGAAATC ATcGGTATga AaGACggTTC AAA
181


 


S. capitis capitis
AG TtGGTGAAGA AgTtGAAATC ATcGGTATCC AcGAaACTTC TAA
182


 


M. caseolyticus
AG TtGGTGAAGA AgTtGAAATC ATTGGTtTaa cTGAagaacC AAA
183


 


S. cohnii
AG TCGGTGAAGA AgTtGAAATC ATcGGTATgC AaGAagaTTC CAA
184


 


S. epidermidis
AG TtGGTGAAGA AgTtGAAATC ATcGGTATgC AcGAaACTTC TAA
185


 


S. haemolyticus
AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA
186


 


S. haemolyticus
AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGkCACTTC TAA
189


 


S. haemolyticus
AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA
190


 


S. haemolyticus
AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA
188


 


S. hominis
AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA
194


 


S. hominis hominis
AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA
191


 


S. hominis
AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA
193


 


S. hominis
AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA
195


 


S. hominis
AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAtACTTC TAA
196


 


S. lugdunensis
AG TCGGTGAAGA AgTtGAAATt ATTGGTATCC AcGAtACTaC TAA
197


 


S. saprophyticus
AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA
198


 


S. saprophyticus
AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA
200


 


S. saprophyticus
AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA
199


 


S. sciuri sciuri
TG TtGGTGAAGA AgTtGAAATC ATcGGTtTaa cTGAagaaTC TAA
201


 


S. warneri
AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA
187


 


S. warneri
AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA
192


 


S. warneri
AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA
202


 


S. warneri
AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA
203


 


B. subtilis
AG TCGGTGAcGA AgTtGAAATC ATcGGTcTtC AaGAagagag AAA
−2414a


 


E. coli
AG TtGGTGAAGA AgTtGAAATC gTTGGTATCa AaGAgACTca GAA
78


 


L. monocytogenes
AG TtGGTGAcGA AgTaGAAgTt ATcGGTATCg AaGAagaaag AAA
2415


 


Selected sequences
    CGGTGAAGA AATCGAAATC A (S. saprophyticus)
599


for species-specific
        (S.haemolyticus) ATTGGTATCC ATGACACTTC
594


hybridization probes





The sequence numbering refers to the Staphylococcus aureus tuf gene fragment (SEQ ID NO. 179).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


aThis sequence was obtained from Genbank accession #Z99104.













TABLE 53







Strategy for the selection of Staphylococcus aureus-


specific and of Staphylococcus epidermidis-specific


hybridization probes from tuf sequences.










521                      547   592                      617
SEQ ID NO.:





S. aureus
TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT
179


 


S. aureus
TACACCACA TACTGAATTC AAAGCAG...TTCTTCtC~ ~~~~~~~~~~ ~~~~~~~
178


 


S. aureus
TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT
176


 


S. aureus
TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT
177


 


S. aureus aureus
TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT
180


 


S. auricularis
TACACCACA cACTaAATTC ActGCAG...TTCTTCtCT AACTAcCGtC CACAATT
181


 


S. capitis capitis
CACACCACA cACTaAATTC AAAGCGG...TTCTTCAgT AACTACCGCC CACAATT
182


 


M. caseoiyticus
TACtCCACA TACTaAATTC AAAGCTG...TTCTTCACT AACTAcCGCC CtCAGTT
183


 


S. cohnii
TACACCACA cACaaAcTTt AAAGCAG...TTCTTcAgT AACTATCGCC CACAATT
184


 


S. epidermidis
TACACCACA cACaaAATTC AAAGCTG...TTCTTCACT AACTATCGCC CACAATT
185


 


S. haemolyticus
CACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtC CACAATT
186


 


S. haemolyticus
CACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtC CACAATT
189


 


S. haemolyticus
CACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtC CACAATT
190


 


S. haemolyticus
TACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtC CACAATT
188


 


S. hominis
CACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtC CACAATT
195


 


S. hominis
TACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtC CACAATT
196


 


S. hominis hominis
TACACCtCA cACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtC CACAATT
191


 


S. hominis
TACACCtCA cACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtC CACAATT
193


 


S. hominis
TACACCtCA CACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtC CACAATT
194


 


S. lugdunensis
TACACCtCA CACTaAATTt AAAGCTG...TTCTTCtCa AACTAcCGCC CACAATT
197


 


S. saprophyticus
TACACCACA TACaaAATTC AAAGCAG...TTCTTCACT AACTAeCGCC CACAATT
198


 


S. saprophyticus
TACACCACA TACaaAATTC AAAGCAG...TTCTTCACT AACTAcCGCC CACAATT
199


 


S. saprophyticus
TACACCACA TACaaAATTC AAAGCAG...TTCTTCACT AACTAcCGCC CACAATT
200


 


S. sciuri sciuri
CACACCtCA CACTaAATTC AAAGCTG...TTCTTCACa AACTAcCGCC CACAATT
201


 


S. warneri
TACACCACA TACaaAATTC AAAGCGG...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~
192


 


S. warneri
TACACCACA TACaaAATTC AAACCGG...TTCTTCAgT AACTAcCGCC CACAATT
187


 


S. warneri
TACACCACA TACaaAATTC AAAGCAG...TTCTTCAgT AACTAcCGCC CACAATT
202


 


S. warneri
TACACCACA TACaaAATTC AAAGCAG...TTCTTCAgT AACTAcCGCC CACAATT
203


 


B. subtilis
CACtCCACA cAgCaAATTC AAAGCTG...TTCTTCtCT AACTAcCGtC CtCAGTT
−a2416


 


E. coli
CAAgCCgCA CACcaAgTTC gAAtCTG...TTCTTCAaa ggCTAcCGtC CgCAGTT
78


 


L. monocytogenes
TACtCCACA CACTaAcTTC AAAGCAG...TTCTTCAac AACTAcCGCC CACAATT
2417


 


Selected sequences
   ACCACA TACTGAATTC AAAG (S. aureus)
585


for species-specific
                  (S.epidermidis) TTCACT AACTATCGCC CACA
593


hybridization




probes





The sequence numbering refers to the Staphylococcus aureus tuf gene fragment (SEQ ID NO. 179).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lower-case letters.


“~” indicate incomplete sequence data.


Dots indicate gaps in the sequences displayed.


aThis sequence was obtained from Genbank accession #Z99104.













TABLE 54







Strategy for the selection of the


Staphylococcus hominis-specific


hybridization probe from tuf sequences.










358                       383
SEQ ID NO.:





S. aureus
ATC ATcGGTtTac AtGAcACaTC TAA
179


 


S. aureus
ATC ATcGGTtTac AtGAcACaTC TAA
176


 


S. aureus
ATC ATcGGTtTac AtGAcACaTC TAA
177


 


S. aureus
ATC ATcGGTtTac AtGAcACaTC TAA
178


 


S. aureus aureus
ATC ATcGGTtTac AtGAcACaTC TAA
180


 


S. auricularis
ATC ATcGGTATgA AAGAcggTTC AAA
181


 


S. capitis capitis
ATC ATcGGTATCc AcGAAACTTC TAA
182


 


M. caseolyticus
ATC ATTGGTtTaA ctGAAgaacC AAA
183


 


S. cohnii
ATC ATcGGTATgc AAGAAgaTTC CAA
184


 


S. epidermidis
ATC ATcGGTATgc AcGAAACTTC TAA
185


 


S. haemolyticus
ATC ATTGGTATCc AtGAcACTTC TAA
186


 


S. haemolyticus
ATC ATTGGTATCc AtGAcACTTC TAA
189


 


5: haemolyticus
ATC ATTGGTATCc AtGAcACTTC TAA
190


 


S. haemolyticus
ATT ATTGGTATCA AAGAAACTTC TAA
188


 


S. hominis
ATT ATTGGTATCA AAGAtACTTC TAA
196


 


S. hominis
ATT ATTGGTATCA AAGAAACTTC TAA
194


 


S. hominis hominis
ATT ATTGGTATCA AAGAAACTTC TAA
191


 


S. hominis
ATT ATTGGTATCA AAGAAACTTC TAA
193


 


S. hominis
ATT ATTGGTATCA AAGAAACTTC TAA
195


 


S. lugdunensis
ATT ATTGGTATCc AcGAtACTaC TAA
197


 


S. saprophyticus
ATC ATcGGTATgc AAGAAgaaTC CAA
198


 


S. saprophyticus
ATC ATcGGTATgc AAGAAgaaTC CAA
200


 


S. saprophyticus
ATC ATcGGTATgc AAGAAgaaTC CAA
199


 


S. sciuri sciuri
ATC ATcGGTtTaA ctGAAgaaTC TAA
201


 


S. warneri
ATC ATcGGTtTac AtGAcACTTC TAA
187


 


S. warneri
ATC ATcGGTtTac AtGAcACTTC TAA
192


 


S. warneri
ATC ATcGGTtTac AtGAcACTTC TAA
202


 


S. warneri
ATC ATcGGTtTac AtGAcACTTC TAA
203


 


B. subtilis
ATC ATcGGTcTtc AAGAAgagag AAA
2418−a


 


E. coli
ATC gTTGGTATCA AAGAgACTCa GAA
78


 


L. monocytogenes
GTT ATcGGTATCg AAGAAgaaag aaa
2419


 


Selected sequence for
    ATTGGTATCA AAGAAACTTC
597


species-specific




hybridization probe





The sequence numbering refers to the Staphylococcus aureus tuf gene fragment (SEQ ID NO. 179).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequences displayed.


aThis sequence was obtained from Genbank accession #Z99104.













TABLE 55







Strategy for the selection of the Enterococcus-


specific amplification primers from tuf sequences.












SEQ ID




270                         298   556                        582
NO.:
Accession #:













E. avium
TAGAATTAAT GGCTGCTGTT GACGAATAT...TGAA GATATCCAAC GTGGACAAGT ATT
2420
—


 


E. casseliflavus
TGGAATTAAT GGCTGCAGTT GACGAATAC...TGAA CACATCCAAC GTGGACAAGT ATT
58
—


 


E. cecorum
TAGAATTAAT GGCTCCAGTT GACGAATAC...TGAA GATATCCAAC GTGGtCAACT ATT
59
—


 


E. dispar
TAGAATTAAT GGCTGCAGTT GACGAATAT...TGAA CATATCCAAC GTGGtCAAGT ATT
60
—


 


E. durans
TTGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT TTT
61
—


 


E. flavescens
TGGAATTAAT GGCTGCAGTT GACCAATAC...TGAA GACATCCAAC GTGGACAAGT ATT
65
—


 


E. faecium
TTGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT TTT
608
—


 


E. faecalis
TAGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GATATCGAAC GTGGACAAGT ATT
607
—


 


E. gallinarum
TGGAATTgAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT
609
—


 


E. hirae
TTGAATTgAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT TTT
67
—


 


E. mundtii
TTGAATTgAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC CTGGtCAAGT TTT
68
—


 


E. pseudoavium
TAGAATTAAT GSCTCCTGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT
69
—


 


E. raffinosus
TAGAATTAAT GGCTGCTGTT GATGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT
70
—


 


E. saccharolyticus
TCGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT ATT
71
—


 


E. solitarius
TGGAcTTAAT GGaTGCAGTT GATGAcTAC...TGAt GATATCGAAC GTCGtCAAGT ATT
72
—


 


E. coli
TGGAAcTggc tGgcttccTg GATtctTAY...TGAA GAaATCGAAC GTGGtCAgGT ACT
78
—


 


B. cepacia
TGAgccTggc cGacGcgcTg GACaCgTAC...TGAA GACgTgGAgC GTGGcCAgCT TCT
16
—


 


B. fragilis
TGGAAcTgAT GGaaGCTGTT GATactTGG...GAAc GAaATCaAAC CTGGtatgGT TCT
−2421 
M22247


 


B. subtilis
TCGAAcTtAT GGaTGCgGTT GATGAgTAC...TGAA GAaATCCAAC GTGGtCAAGT ACT
−2422 
Z99104


 


C. diphtheriae
TCGAccTcAT GcagGCTtgc KATGAtTCC...CGAA GACgTtGAgC GTGGcCAgGT TGT
662
—


 


C. trachomatis
GAGAgcTAAT GcaaGCcGTc GATGAtAAT...GAAc GATgTgGAAa GaGGAatgGT TGT
22
—


 


G. vaginalis
AGGAAcTcAT GaagGCTGTT GACGAgTAC...TACc GACgTtCAgC GTGGtCAgGT TGT
2423
—


 


S. aureus
TAGAATTAAT GGaaGCTGTa GATactTAC...TGAA GACgTaCAAC GTGGtCAAGT ATT
179
—


 


S. pneumoniae
TGGAATTgAT GaacaCAGTT GATGAgTAT...TGAt CAaATCGAAC GTGGACAACT TAT
2424
—


 


A. adiacens
TAGAATTAAT GGCTCCTGTT GACGAATAC...TGAA aACATCGAAC GTGGACAAGT TCT
2425
—


 


G. haemolysans
TCGAATTAAT GGaaaCAGTT GACGAATAC...TGAA GACATCGAAC GTGGACAAGT TTT
87
—


 


G. morbillorum
TCGAATTAAT GGaaaCAGTT GACGAgTAC...TGAA GATATCGAAC GTGGACAAGT TTT
88
—


 


Selected sequence
   AATTAAT GGCTGCWGTT GAYGAA
1137



for amplification





primer





 


Selected sequence
                                     A GAYATCSAAC GTGGACAAGT
1136



for amplification





primerb





The sequence numbering refers to the Enterococcus durans tuf gene fragment (SEQ ID NO. 61).


Nucleotides in capitals are identical to the selected or match those sequences.


Mismatches are indicated bv lower-case letters.


Dots indicate gaps in the sepuences displayed.


“Y” “W” and “S” designate nucleotide positions which are degenerated.


“Y” stands for C or T;


“W” stands for A or T;


“S” stands for C or G;


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, c, G or T.


bThis sequence is the reverse-complement of the selected primer.













TABLE 56







Strategy for the selection of the Enterococcus faecalis-specific


hybridization probe, of the Enterococcus faecium-specific hybridization


probe and of the Enterococcus casseliflavus-flavescens-gallinarum


group-specific hybridization probe from tuf sequences.











395
SEQ ID NO.:
Accession #:













E. avium
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa
2426
—


 


E. casseliflavus
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT
58
—


 


E. cecorum
GTTGA ACGTGGaCAA GTaCGtGTTG GTGACGAAGT TGAAaTaGTT
59
—


 


E. dispar
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa
60
—


 


E. durans
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAtaTcGTT
61
—


 


E. faecalis
GTTGA ACGTGGTGAA GTTCGCGTTG GTGACGAAGT TGAAaTcCTT
62
—


 


E. faecium
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAGTTGTT
608
—


 


E. flavescens
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT
65
—


 


E. gallinarum
GTTGA ACGTGGacAA GTTCGCGTTG GTGATGAAGT aGAAaTcGTT
609
—


 


E. hirae
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAtaTcGTT
67
—


 


E. mundtii
GTTGA ACGTGGacAA GTTCGtGTTG GTGACGttaT cGAtaTcGTT
68
—


 


E. pseudoavium
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa
69
—


 


E. raffinosus
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa
70
—


 


E. saccharolyticus
GTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAAaTcGTT
71
—


 


E. solitarius
GTTGA ACGcGGgact aTcaaaGTCG GCGATGAAGT TGAcaTTaTT
72
—


 


C. diphtheriae
GTTGA gCGTGGctcc cTgaagGTCA ACGAGGAcGT cGAgaTcaTc
662
—


 


G. vaginalis
GTTGA gCGTGGTaAg cTcCcaATCA ACACCCcAGT TGAgaTcGTT
135a
—


 


B. cepacia
GTCGA gCGcGGcatc GTgaagGTCG GCGAAGAAaT cGAAaTcGTc
16
—


 


S. aureus
GTTGA ACGTGGTcAA aTcaaaGTTG GTGAAGAAGT TGAAaTcaTc
179
—


 


B. subtilis
GTAGA ACGaGGacAA GTTaaaGTCG GTGACGAAGT TGAAaTcaTc
−2427
Z99104


 


S. pneumoniae
ATCGA cCGTGGTatc GTTaaaGTCA ACGACGAAaT cGAAaTcGTT
2428
—


 


E. coli
GTAGA ACGcGGTatc aTcaaaGTTG GTGAAGAAGT TGAAaTcGTT
78
—


 


B. fragilis
ATCGA AacTGGTGtt aTcCatGTAG GTGATGAAaT cGAAaTccTc
−2429
M22247


 


C. trachomatis
ATTGA gCGTGGaatt GTTaaaGTTT CCGATAAAGT TcAgtTgGTc
22
—


 


Selected sequences
   GA ACGTGGTGAA GTTCGC (E. faecalis)
1174



for species-specific
                                  AAGT TGAAGTTGTT




or group-specific





hybridization probes





 













      448...526                    549
SEQ ID NO.:
Accession #:






E. avium
GGTATcGCT...CATc GGTGCtTTCt TACGTGGTGT
131A
—


 



E. casseliflavus
GGTATTGCT...CATT GGTGCATTGC TACGTGGTGT
58
—


 



E. cecorum
GGTATcCAT...CATc GGTCCATTat TACGTGGTGT
59
—


 



E. dispar
GGTATcGCT...CATT GGTGCATTat TACGTGGTGT
60
—


 



E. durans
GGTATcCCA...CATT GGTGCtTTaC TACGTCGTGT
61
—


 



E. faecalis
GGTATTAAA...CTTc GGTGCtTTat TACGTGGTGT
62
—


 



E. faecium
GGTATTGCT...CATT GGTGCtTTaC TACGTGGTGT
608
—


 



E. flavescens
GGTATTGCT...CATT GGTGCATTGC TACGTGGGGT
65
—


 



E. gallinarum
GGTATTGCT...CATT GGTGCATTGC TACGTGGGGT
609
—


 



E. hirae
GGTATcGCA...CATT GGTGCtTTaC TACGTGGTGT
67
—


 



E. mundtii
GGTATcGCA...CATT GGTGCgTTaC TACGTGGTGT
68
—


 



E. pseudoavium
GGTATCGCT...CATc GGTGCATTat TACGTGGTGT
69
—


 



E. raffinosus
GGTATTGCT...CATT GGTGCATTat TACGTGGTGT
70
—


 



E. saccharolyticus
GGTATcGAC...CATc GGTGCtTTat TACGTGGGGT
71
—


 



E. solitarius
GGTATTGAT...CATT GGTaCtTTGt TACGTGGTGT
72
—


 



C. diphtheriae
GGTATcCGC...CTGT GGTctgcTtC TcCGTGGCGT
662
—


 



G. vaginalis
GGTtTgCGC...CACT GGTcttcTtC TcCGcCGTAT
135a
—


 



B. cepacia
GGTATCAAG...CGTT GGTatccTGC TgCGcGGCAC
16
—


 



S. aureus
GGTtTaCAT...CATT GGTGCATTat TACGTGGTGT
179
—


 



B. subtilis
GGTcTTCAA...CATT GGTGCccTtC TtCGcGGTGT
−2427
Z99104


 



S. pneumoniae
GGTATcAAA...CGTa GGTGtccTtC TtCGTGGTGT
2428
—


 



E. coli
GGTATcAAA...CGTa GGTGttcTGC TgCGTGGTAT
78
—


 



B. fragilis
GGTtTgGGT...CGTa GGTctgTTGC TtCGTGGTGT
−2429
M22247


 



C. trachomatis
GGTcTTAGA...CGTT GGattgcTcC TcaGaGGTAT
22
—


 



Selected sequences






for species-specific






or group-specific
GGTATT (E. faecium)
602




hybridization probes
               T GGTGCATTGC TACGTGG
1122





The sequence numbering refers to the Enterococcus faecium tuf gene fragments (SEQ ID NO. 608).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequences displayed.













TABLE 57







Strategy for the selection of primers for the


identification of platelets contaminants from tuf sequences.












SEQ ID
Accession



467                     495        689                          717
NO.:
#:













B. cereus
GTA ACTGGTGTaG AGATGTTCCG TAAACT...C AGTTCTACTT CCGTACAACT GACGTAAC
7
—


 


B. subtilis
GTT ACaGGTGTTG AAATGTTCCG TAAGCT...C AGTTCTACTT CCGTACAACT GACGTAAC
−2430
Z99104


 


E. cloacae
TGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GACGTGAC
54
—


 


E. coli
TGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACTACT GACGTGAC
78
—


 


K. oxytoca
TGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GACGTGAC
100
—


 


K. pneumoniae
TGT ACTGGCGTTG AAATGTTCCG CAAAGT...C AGTTCTACTT CCGTACTACT GACGTGAC
103
—


 


P. aeruginosa
TGG ACcGGCGTTG AAATGTTCCG CAAGCT...C AGTTCTACTT CCGTACCACT GACGTGAC
153
—


 


S. agalactiae
GTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT CCGTACAACT GACGTAAC
209
—


 


S. aureus
GTT ACaGGTGTTG AAATGTTCCG TAAATT...C AATTCTATTT CCGTACTACT GACGTAAC
2431
—


 


S. choleraesuis
TGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACTACT GACGTGAC
159
—


 


S. epidermidis
GTT ACTGGTGTaG AAATGTTCCG TAAATT...C AATTCTATTT CCGTACTACT GACGTAAC
611
—


 


S. marcescens
TGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACCACT GACGTGAC
168
—


 


S. mutans
GTT ACTGGTGTTG AAATGTTCCG TAAACT...C AATTCTACTT CCGTACAACT GACGTAAC
224
—


 


S. pyogenes
GTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT CCGTACAACT GACGTAAC
−2433
U40453


 


S. salivarius
GTT ACTGGTGTTG AAATGTTCCG TAAACA...C AGTTCTACTT CCGTACAACT GACGTAAC
146a
—


 


S. sanguinis
GTT ACTGGTGTTG AAATGTTCCG TAAACA...C AGTTCTACTT CCGTACAACT GACGTTAC
227
—


 


Y. enterocolitica
TGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GAtGTAAC
235
—


 


Selected sequence
    ACTGGYGTTG AIATGTTCCG YAA
636



for amplification





primer





 


Selected sequence
                                       TTCTAYTT CCGTACIACT GACGT
637



for amplification





primerb





The sequence numbering refers to the E. coli tuf gene fragment (SEQ ID NO. 78).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lower-case letters.


Dots indicate gaps in the sequences displayed.


“R” “Y” “N” “K” “WI” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“M” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.


bThis sequence is the reversecomplement of the selected primer.













TABLE 58







Strategy for the selection of the universal amplification primers from atpD sequences.











616                                        657   781
SEQ ID NO.
ACCESSION #:





C. glutamicum
GTGTTCGGTC AGATGGATGA GCCACCAGGA GTCCGTATG CGC...CGTATg
−2434
X76875


 


M. tuberculosis
GTATTCGGAC AGATGGACGA GCCGCCGGGC aCCCGTATG CGT...CGGATg
−2435
Z73419


 


E. faecalis
GTGTTCGGAC AAATGAACGA ACCACCAGGT GCTCGGATG CGG...CGTATg
291
—


 


S. agalactiae
GTCTTTGGTC AAATGAATGA ACCACCAGGA GCACGTATG CGT...CGTATg
380
—


 


B. subtilis
GTATTCGGAC AAATGAACGA GCCGCCGGGC GCACGTATG CGT...CGTATg
−2436
Z28592


 


L. monocytogenes
GTATTCGGTC AAATGAACGA GCCACCAGGT GCGCGTATG CGT...CGTATg
324
—


 


S. aureus
GTATTCGGGC AAATGAATGA GCCACCTGGT GCACGTATG CGT...CGTATg
366
—


 


A. baumannii
GTCTACGGTC AGATGAACGA GCCACCAGGT aaCCGTtTa CGC...CGTATg
243
—


 


N. gonorrhoeae
GTGTATGGCC AAATGAACGA ACCTCCAGGC aaCCGTcTG CGC...CGTATg
−2437
Genome project


 


C. freundii
GTATATGGCC AGATGAACGA GCCGCCTGGA aaCCGTcTG CGT...CGTATg
264
—


 


E. cloacae
GTTTACGGCC AGATGAACGA GCCACCAGGA aaCCGTcTG CGC...CGTATg
284
—


 


E. coli
GTGTATGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg
669
V00267


 


S. typhimurium
GTGTATGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg
351
—


 


K. pneumoniae
GTGTACGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg
317
—


 


S. marcescens
GTTTACGGCC AGATGAACGA GCCACCAGGT aaCCGTcTG CGC...CGTATg
357
—


 


Y. enterocolitica
GTTTATGGCC AAATGAATGA GCCACCAGGT aaCCGTcTG CGC...CGTATg
393
—


 


B. cepacia
GTGTACGGCC AGATGAACGA GCCGCCGGGC aaCCGTcTG CGC...CGTATg
−2438
X76877


 


H. influenzae
GTTTATGGTC AAATGAACGA GCCACCAGGT aaCCGTtTa CGT...CGTATg
−2439
U32730


 


M. pneumoniae
GTGTTTGGTC AGATGAACGA ACCCCCAGGA GCACGGATG CGG...CGGATg
−2440
U43738


 


H. pylon
TGCTATGGGC AAATGAATGA GCCACCAGGT GCAAGGAat CGC...CGTATC
670
V00267


 


B. fragilis
GTGTTCGGAC AGATGAACGA ACCTCCTGGA GCACGTgct TCA...CGTATg
−2441
M22247


 


Selected sequences
         C ARATGRAYCA RCCICCIGGI GYIMGIATG
562



for universal
 TAYGGIC APATGAAYGA RCCICCIGGI AA
564



primers





 


Selected sequences
                                                    ATH
565



for universal
                                                    ATG
563



primersa
















                         812
SEQ ID NO
ACCESSION #






C. glutamicum
CCTTCCGCCG TGGGTTACCA GCCAAC
−2434
X76875


 



M. tuberculosis
CCGTCGGCCG TGGGATACCA GCCCAC
−2435
Z73419


 



E. faecalis
CCTTCTGCCG TTGGTTACCA ACCAAC
291
—


 



S. agalactiae
CCTTCAGCCG TTGGTTATCA ACCAAC
380
—


 



B. subtilis
CCTTCAGCGG TTGGTTATCA GCCGAC
−2436
Z28592


 



L. monocytogenes
CCATCTGCGG TAGGTTACCA ACCAAC
324
—


 



S. aureus
CCTTCTGCAG TAGGTTACCA ACCAAC
366
—


 



A. baumannii
CCATCTGCGG TAGGTTACCA ACCTAC
243
—


 



N. gonorrhoeae
CCTTCTGCAG TGGGTTACCA ACCGAC
−2437
Genome project


 



C. freundii
CCATCAGCGG TAGGCTACCA GCCGAC
264
—


 



E. cloacae
CCTTCAGCGG TAGGTTATCA GCCTAC
284
—


 



E. coli
CCTTCAGCGG TAGGTTATCA GCCGAC
669
V00267


 



S. typhimurium
CCTTCCGCAG TAGGTTACCA GCCGAC
351
—


 



K. pneumoniae
CCTTCAGCGG TAGGTTATCA GCCGAC
317
—


 



S. marcescens
CCATCCGCGG TACGTTATCA GCCAAC
357
—


 



Y. enterocolitica
CCATCTGCCG TAGGTTACCA GCCAAC
393
—


 



B. cepacia
CCGTCGGCAG TGGGCTATCA GCCGAC
−2438
X76877


 



H. influenzae
CCATCCGCGG TAGGTTACCA ACCGAC
−2439
U32730


 



M. pneumoniae
CCATCAGCCG TGGGTTACCA ACCAAC
−2440
U43738


 



H. pylon
CCTTCAGCGG TGGGGTATCA GCCCAC
670
V00267


 



B. fragilis
CCTTCTGCGG TAGGTTATCA ACCTAC
−2441
M22247


 



Selected sequences






for universal






primers





 



Selected sequences
CCITCIGCIG TIGGITAYCA RCC
565




for universal
CCITCIGCIG TIGGITAYCA RCC
563




primersa





The sequence numbering refers to the Escherichia coli atpD gene fragment (SEQ ID NO. 669).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches for SEQ ID NOs. 562 and 565 are indicated by lowercase letters.


Mismatches for SEQ ID NOs. 564 and 563 are indicated by underlined nucleotides.


Dots indicate gaps in the sequences displayed.


“R” “Y” “M” “K” “W” and “S” letters designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“N” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“H” stands for A, C or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.


aThese sequences are the reverse-complement off the selected primers.













TABLE 59







Specific and ubiquitous primers for


nucleic acid amplification (recA sequences).











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position






Universal primers (recA)




919 
5′-GGI CCI GAR TCI TMI GGI AAR AC
918a
437-459


920b
5′-TCI CCV ATI TCI CCI TCI AIY TC
918a
701-723


 


921 
5′-TIY RTI GAY GCI GAR CAI GC
918a
515-534


922b
5′-TAR AAY TTI ARI GCI YKI CCI CC
918a
872-894


 



Sequencing primers (recA)




1605 
5′-ATY ATY GAA RTI TAY GCI CC
1704a
220-239


1606 
5′-CCR AAC ATI AYI CCI ACT TTT TC
1704a
628-650


 



Universal primers (rad51)




935 
5′-GGI AAR WSI CAR YTI TGY CAY AC
939a
568-590


936b
5′-TCI SIY TCI GGI ARR CAl GG
939a
1126-1145


 



Universal primers (dmc1)




937 
5′-ATI ACI GAR GYI TTY GGI GAR TT
940a
1038-1060


938b
5′-CYI GTI GYI SWI GCR TGI GC
940a
1554-1573





aSequences from databases.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.













TABLE 60







Specific and ubiquitous primers for


nucleic acid amplification (speA sequences).











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Bacterial species:
Streptococcus pyogenes




994 
5′-TGG ACT AAC AAT CTC GCA AGA GG
993a
60-82


995b
5′-ACA TTC TCG TGA GTA ACA GGG T
993a
173-194


 


996 
5′-ACA AAT CAT GAA GGG AAT CAT TTA G
993a
400-424


997b
5′-CTA ATT CTT GAG CAG TTA CCA TT
993a
504-526


 


998 
5′-GGA GGG GTA ACA AAT CAT GAA GG
993a
391-413


997b
5′-CTA ATT CTT GAG CAG TTA CCA TT
993a
504-526





aSequence from databases.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.













TABLE 61







First strategy for the selection of Streptococcus pyogenes-specific


amplification primers from speA sequences.











ACCESSION #
57                        85       170                         197
SEQ ID NO.:













speA
X61573
CCTT GGgCTAACAA cCTCaCAAGA aGTAT...GTGAtCCT.GT cgtTCAtGAG AATGTAAA
−2442


 


speA
AF029051
~~~~ GGgCTAACAA cCTCaCAAGA aCTAT...GTGAtCCT.GT cgtTCAtGAG AATGTAAA
−2443


 


speA
X61571
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2444


 


speA
X61570
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2445


 


speA
X61568
TCTT GGACTAACAA TCTCCCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2446


 


speA
X61569
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2447


 


speA
X61572
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2448


 


speA
X61560
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2449


 


speA
U40453
TCTT CGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
993


 


speA
X61554
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2450


 


speA
X61557
TCTT CGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCAAGAG AATGTGAA
−2451


 


speA
X61559
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2452


 


speA
X61558
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACCAG AATGTGAA
−2453


 


speA
X61556
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2454


 


speA
X61555
TCTT CGACTAACAA TCTCCCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2455


 


speA
X61560
TCTT CGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2456


 


speA
X61561
TCTT CGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2457


 


speA
X62566
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2458


 


speA
X61567
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2459


 


speA
X61562
TCTT GGACTAACAA TCTCCCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2460


 


speA
X61563
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2461


 


speA
X61564
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2462


 


speA
X61565
TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2463


 


speA
AF055698
~~~~ CCACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA
−2464


 


speA
X039298
TCTT GGACTAACAA TCTtGCcAaA aGGTA...GTGACCCTGGT TACTCACGAG AATGTGAA
−2465


 









Selected sequence
   T GGACTAACAA TCTCGCAAGA CC
994


for species-




specific primer




 


Selected sequence
                                      ACCCT.GT TACTCACGAC AATGT
995


for species-




specific primerb





The sequence iiumbering refers to the Streptococcus pyogenes speA gene fragment (SEQ ID NO. 993).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


“~” indicate incomplete sequence data.


Dots indicate gap in the sequences displayed.


aThe extra G nucleotide introducing a gap in the sequence is probably a sequencing error.


bThis sequence is the reverse-complement of the selected primer.













TABLE 62







Second strategy for the selection of Streptococcus pyogenes-specific


amplification primers from speA sequences.













SEQ ID



Accession #
388                               427       501                         529
NO.:













speA
X61573
TA TCGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2466


 


speA
AF029051
TA TGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2467


 


speA
X61571
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2468


 


speA
X61570
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2469


 


speA
X61568
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2470


 


speA
X61569
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2471


 


speA
X61572
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2472


 


speA
X61560
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2473


 


speA
U40453
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
993


 


speA
X61554
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2474


 


speA
X61557
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2475


 


speA
X61559
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTACAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2476


 


speA
X61558
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2477


 


speA
X61556
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTCCTCAA GAATTAGACT
−2478


 


speA
X61555
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2479


 


speA
X61560
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2480


 


speA
X61561
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2481


 


speA
X61566
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2482


 


speA
X61567
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2483


 


speA
X61562
TA CGGAGCGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATCGT AACTGCTCAA GAATTAGACT
−2484


 


speA
X61563
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2485


 


speA
X61564
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2486


 


speA
X61565
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2487


 


speA
AF055698
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT
−2488


 


speA
X03929
TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAG.CT
−2489


 









Selected
    GGAGGGGTA ACAAATCATG AAGG
998


sequences for
              ACAAATCATG AAGGGAATCA TTTAG
996


species-specific




primers




 


Selected
                                                      AATGGT AACTGCTCAA GAATTAG
997


sequence for




species-specific




primersa





The sequence numbering refers to the Streptococcus pyogenes speA gene fragment (SEQ ID NO. 993).


Dots indicate gaps in the sequences displayed.


aThis sequence is the reverse-complement of the selected primer.













TABLE 63







Strategy for the selection of Streptococcus pyogenes-specific


amplification primers from tuf sequences.










140                                              186   619
SEQ ID NO.:












S. anginosus
A AGTTGACtTg GTTGACGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt
211


 


S. bovis
A AGTTGACCTT GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
212


 


S. dysgalactiae
A AATTGACCTT GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
217


 


S. pyogenes
A AGTTGACCTT GTTGATGACG AAGAGTTGCT TGAATTAGTT GAGATG...CC AAGTTCAATC
1002


 


S. agalactiae
A AGTTGACCTT GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
144a


 


S. oralis
A AATTGACtTg GTAGAcGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
985


 


S. pneumoniae
A AGTTGACtTg GTTGAcGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
145a


 


S. cristatus
A GATCGACtTg GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
215


 


S. mitis
A GATCGACtTg GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
982


 


S. gordonii
A AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAGTTgGTT GAaATG...CC AgGTTCAATC
200


 


S. sanguinis
A AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
227


 


S. parasanguinis
A AGTTGACtTg GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC
225


 


S. salivarius
A AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC TgGTTCAATC
146a


 


S. vestibularis
A AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC TgGTTCAATC
231


 


S. suis
A AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAgTTgGTT GAaATG...CC AgGTTCtATC
229


 


S. mutans
A AGTTGAttTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt
224


 


S. ratti
A GGTTGACtTg GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt
226


 


S. macacae
A AGTTGACtTa GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGATCAATt
222


 


S. cricetus
A GGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC TgGTTCAATC
214


 


E. faecalis
A AATgGAtaTg GTTGATGACG AAGAaTTatT aGAATTAGTa GAaATG...CC AgcTaCAATC
607


 


S. aureus
A AGTTGACaTg GTTGAcGAtG AAGAaTTatT aGAATTAGTa GAaATG...CC TgGTTCAATt
176


 


B. cereus
A ATgcGACaTg GTaGATGACG AAGAaTTatT aGAATTAGTa GAaATG...AG CgGTTCtgTa
7


 


E. coli
A ATgcGACaTg GTTGATGACG AAGAGcTGCT gGAAcTgGTT GAaATG...CC GgGCaCcATC
78


 


Selected
    TTGACCTT GTTGkTGACG AAGAG
999


sequences for
                        AAGAGTTGCT TGAATTAGTT GAG
1001


species-specific




primers




 


Selected
                                                          AGTTCAATC
1000


sequence for




species-specific




primerb















               647
SEQ ID NO.:






S. anginosus
cAtCCACACA CTAAATT
211


 



S. bovis
CACCCACACA CTAAATT
212


 



S. dysgalactiae
AACCCACACA CTAAATT
217


 



S. pyogenes
AACCCACACA CTAAATT
1002


 



S. agalactiae
AACCCACACA CTAAATT
144a


 



S. oralis
AACCCACACA CTAAATT
985


 



S. pneumoniae
AACCCACACA CTAAATT
145a


 



S. cristatus
AACCCACACA CTAAATT
215


 



S. mitis
AACCCACACA CTAAATT
982


 



S. gordonii
AACCCACACA CTAAATT
200


 



S. sanguinis
AACCCACACA CTAAATT
227


 



S. parasanguinis
AACCCACACA CTAAATT
225


 



S. salivarius
AACCCACACA CTAAATT
146a


 



S. vestibularis
AACCCACACA CTAAATT
231


 



S. suis
AACCCACACA CTAAATT
229


 



S. mutans
cACCCACAcA CTAAATT
224


 



S. ratti
CAtCCgCAcA CTAAATT
226


 



S. macacae
cAtCCACAcA CTAAATT
222


 



S. cricetus
cAtCCACACA CTAAATT
214


 



E. faecalis
ActCCACACA CaAAATT
607


 



S. aureus
AcaCCACACA CTgAATT
176


 



B. cereus
AAagCtCACg CTAAATT
7


 



E. coli
AAgCCgCACA CcAAGTT
78


 



Selected





sequences for





species-specific





primers




 



Selected
AACCCACACA CTAA
1000



sequence for





species-specific





primerb





The sequence numbering refers to the Streptococcus pyogenes tuf gene fragment (SEQ ID NO. 1002).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequences displayed.


aThe SEG ID NO. refers to previous patent publication WO98/20157.


bThis sequence is the reverse-complement of the selected primer.













TABLE 64







Strategy for the selection stx1-specific amplification primers and hybridization probe.











Accession #
230                               263   343
SEQ ID NO.:













stx1
M19473a
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
−2490


 


stx1
M16625
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
−2491


 


stx1
D417358
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
−2492


 


stx1
Z36900
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
−2493


 


stx1
L04539
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
−2494


 


stx1
M19437
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
−2495


 


stx1
M24352
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
−2496


 


stx1
X07903
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
−2497


 


stx1
Z36899
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
−2498


 


stx1
Z36901
TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG
1076


 


stx2
X61283
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
−2499


 


stx2
L11079
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
−2500


 


stx2
M21534
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
−2501


 


stx2
M36727
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
−2502


 


stx2
X81415
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
−2503


 


stx2
X81416
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
−2504


 


stx2
X81417
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
−2505


 


stx2
X81418
TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa
−2506


 


stx2
E03962
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
−2507


 


stx2
E03959
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
−2508


 


stx2
X07865
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
−2509


 


stx2
Y10775
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
−2510


 


stx2
Z37725
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
1077


 


stx2
Z50754
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCAcATa
−2511


 


stx2
X67514
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
−2512


 


stx2
L11078
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
−2513


 


stx2
X65949
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa
−2514


 


stx2
AF043627
TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACaG aTTTtCaGAT TTTgCACATa
−2515


 









Selected sequence
   ATGTC AGAGGGATAG ATCCAGAGGA AGG
1081


for amplification




primer




 


Selected sequence
                                           CG CTTTGCTGAT TTTTCACATG
1084


for hybridization




probe




 


Selected sequence




for amplification




primera
















Accession #
     375   391                            421
SEQ ID NO.:






stx1
M19473a
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA
−2490


 



stx1
M16625
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA
−2491


 



stx1
D417358
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA
−2492


 



stx1
Z36900
TTACCTTT...GTTACAT TGTCTaGTGA CAGTAGCTAT ACCA
−2493


 



stx1
L04539
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA
−2494


 



stx1
M19437
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA
−2495


 



stx1
M24352
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA
−2496


 



stx1
X07903
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA
−2497


 



stx1
Z36899
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA
−2498


 



stx1
Z36901
TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA
1076


 



stx2
X61283
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2499


 



stx2
L11079
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2500


 



stx2
M21534
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2501


 



stx2
M36727
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2502


 



stx2
X81415
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2503


 



stx2
X81416
TatCaTTG...ATTtCca TGaCaaagGA CAGcAGtTAT ACCA
−2504


 



stx2
X81417
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2505


 



stx2
X81418
TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2506


 



stx2
E03962
TatCaGTG...GTTtCCa TGaCaacgGA CAGcAGtTAT ACCA
−2507


 



stx2
E03959
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2508


 



stx2
X07865
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2509


 



stx2
Y10775
TatCaGTG...GTTtCca TGaCaacgGA CAGCAGtTAT ACCA
−2510


 



stx2
Z37725
TatCaGTG...GTTtCca TGaCaacgGA CAGCAGtTAT ACCA
1077


 



stx2
Z50754
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2511


 



stx2
X67514
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2512


 



stx2
L11078
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2513


 



stx2
X65949
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2514


 



stx2
AF043627
TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA
−2515


 











Selected sequence





for amplification





primer




 



Selected sequence
TTACC
1084



for hybridization





probe




 



Selected sequence
              ACAT TGTCTGGTGA CAGTAGCTAT A
1080



for amplification





primera





The sequence numbering refers to the Escherichia coil stx1 gene fragment (SEQ ID NO. 1076).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequences displayed.


aThis sequence is the reverse-complement of the selected primer.













TABLE 65







Strategy for the selection of stx2-specific


amplification primers and hybridization probe.











Accession #
543                         570   614                            641
SEQ ID NO.













stx1
M19473
AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A
−2516


 


stx1
M16625
AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTcggC gTtcTTA...A
−2517


 


stx1
M17358
AGCga TgtTaCGgTT TCTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggc gTtcTTA...A
−2518


 


stx1
Z36900
AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A
−2519


 


stx1
L04539
AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A
−2520


 


stx1
M19437
AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTCgaTGAt ctcAgTGggC gTtcTTA...A
−2521


 


stx1
M24352
AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A
−2522


 


stx1
XC7903
AGCga TgtTaCGgTT TCTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTCggC gTtcTTA...A
−2523


 


stx1
Z36899
AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A
−2524


 


stx1
Z36901
AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTtgaTGAt ctcAgTGggC gTtcTTA...A
1076


 


stx2
X61283
AGCAC TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2525


 


stx2
L11079
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2526


 


stx2
M21534
AGGAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G
−2527


 


stx2
M36727
AGGAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G
−2528


 


stx2
U72191
AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G
−2529


 


stx2
X81415
AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G
−2530


 


stx2
X81416
AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G
−2531


 


stx2
X81417
AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G
−2532


 


stx2
X81418
AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G
−2533


 


stx2
E03962
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2534


 


stx2
E03959
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2535


 


stx2
X07865
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2536


 


stx2
Y10775
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2537


 


stx2
Z37725
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
1077


 


stx2
Z50754
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2538


 


stx2
X67514
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2539


 


stx2
L11078
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2540


 


stx2
X65949
AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G
−2541


 


stx2
AF043627
AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G
−2542


 









Selected sequence
   AG TTCTGCGTTT TGTCACTGTC
1078


for amplification




primer




 


Selected sequence
                                     C ACTGTCTGA. ..AACTGCTC CTGT
1085


for hybridization




probe




 


Selected sequence




for amplification




primera
















Accession #
684                    708
SEQ ID NO.:






stx1
M19473
AGgtTgAGtA gCGTcCTgCC tGAC
−2516


 



stx1
M16625
AGgtTgAGtA gCCTcCTgCC tGAC
−2517


 



stx1
M17358
AGgtTgACtA gCGTcCTgCC tGAC
−2518


 



stx1
Z36900
AGgtTgAGtA gTGTcCTgCC tGAT
−2519


 



stx1
L04539
AGgtTgAGtA gTGTcCTgCC tGAT
−2520


 



stx1
M19437
AGgtTgAGtA gTGTcCTgCC tGAC
−2521


 



stx1
M24352
AGgtTgAGtA gTGTcCTgCC tGAC
−2522


 



stx1
XC7903
AGgtTgAGtA gTGTcCTgCC tGAC
−2523


 



stx1
Z36899
AGgtTgAGtA gTGTcCTgCC tGAC
−2524


 



stx1
Z36901
AGgtTgAGtA gTGTcCTgCC tGAC
1076


 



stx2
X61283
CGAATCAGCA ATGTGCTTCC GGAG
−2525


 



stx2
L11079
CGAATCAGCA ATGTGCTTCC GGAG
−2526


 



stx2
M21534
AGAATCAGCA ATGTGCTTCC GGAG
−2527


 



stx2
M36727
AGAATCAGCA ATGTGCTTCC GGAG
−2528


 



stx2
U72191
AGAATCAGCA ATGTGCTTCC GGAG
−2529


 



stx2
X81415
AGAATCAGCA ATGTGCTTCC GGAG
−2530


 



stx2
X81416
AGAATCAGCA ATGTGCTTCC GGAG
−2531


 



stx2
X81417
AGAATCAGCA ATGTGCTTCC GGAG
−2532


 



stx2
X81418
AGAATCAGCA ATGTGCTTCC GGAG
−2533


 



stx2
E03962
CGAATCAGCA ATGTGCTTCC GGAG
−2534


 



stx2
E03959
CGAATCAGCA ATGTGCTTCC GGAG
−2535


 



stx2
X07865
CGAATCAGCA ATGTGCTTCC GGAG
−2536


 



stx2
Y10775
CGAATCAGCA ATGTGCTTCC GGAG
−2537


 



stx2
Z37725
CGAATCAGCA ATGTGCTTCC GGAG
1077


 



stx2
Z50754
CGAATCAGCA ATGTGCTTCC GGAG
−2538


 



stx2
X67514
CGAATCAGCA ATGTGCTTCC GGAG
−2539


 



stx2
L11078
AGAATCAGCA ATGTGCTTCC GGAG
−2540


 



stx2
X65949
AGAATCAGCA ATGTGCTTCC GGAG
−2541


 



stx2
AF043627
AGAATCAGCA ATGTGCTTCC GGAG
−2542


 



Selected sequence






for amplification






primer





 



Selected sequence






for hybridization






probe





 



Selected sequence
  
  AATCAGCA ATGTGCTTCC G
1079



for amplification






primera





The sequence numbering refers to the Escherichia coli stx2 gene fragment (SEQ ID NO. 1077).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatchs are indicated by lowercase letters.


Dots indicate gaps in the sequences displayed.


aThis sequence is the reverse-complement of the selected primer.













TABLE 66







Strategy for the selection of vanA-specific


amplification primers from van sequences.











Accession #
926                        952   1230                     1255
SEQ ID NO.:













vanA
X56895
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG
1139


 


vanA
M97297
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG
1141


 


vanA
—
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG
1051


 


vanA
—
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG
1052


 


vanA
—
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG
1053


 


vanA
—
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG
1054


 


vanA
—
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG
1055


 


vanA
—
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG
1056


 


vanA
—
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGACGTCTAG CCCGTGTGGA TATG
1057


 


vanA
—
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGACGTCTAG CCCGTGTGGA TATG
1049


 


vanA
—
GTCAAT AGCGCGGACG AATTGGACTA C...GT AGACGTCTAG CCCGTGTGGA TATG
1050


 


vanB
U94526
GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT
1117


 


vanB
U94527
GTAAAC AGtaCGGAaG AAcTaaACCC T...GC AGAGGgCTtG CtCGTGTtGA TCTT
−2543


 


vanB
U94528
GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT
−2544


 


vanB
U94529
GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT
−2545


 


vanB
U94530
GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT
−2546


 


vanB
Z83305
GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT
−2547


 


vanB
U81452
GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT
−2548


 


vanB
U35369
GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGACGgCTtG CtCGTGTtGA TCTT
−2549


 


vanB
U72704
GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT
−2550


 


vanB
L06138
CTAAAc AGtaCGGAaG AAcTaaACCC T...GC AGAGGgCTtG CtCGTGTtGA TCTT
−2551


 


vanB
L15304
GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT
−2552


 


vanB
U00456
GTAAAc AGtaCGGAaG AAcTaaACCC T...GC AGAGGgCTtG CtCGTGTtGA TCTT
−2553


 


vanD
AF130997
GTAtgc AagGCaGAaG AAcTGcAgGC A...GC AGAGGatTgG CCCGcaTtGA cCTG
−2554


 


vanE
AF136925
GTAgAa caaaaaagtG AtTTatAtAA A...GC AaAGGatTAG CgaGaaTcGA cTTT
−2555


 









Selected sequence
   AAT AGCGCGGACG AATTGGAC
1090


for amplification




primer




 


Selected sequence
                                     GAGGTCTAG CCCGTGTGGA T
1089


for amplification




primera





The sequence numbering refers to the Enterococcus faecium vanA gene fragment (SEQ ID NO. 1139).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequences


aThis sequence is the reverse-complement of the above selected primer.













TABLE 67







Strategy for the selection of vanB-specific


amplification primers from van sequences.











Accession #
470                       495   608                       633
SEQ ID NO.













vanA
X56895
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1139


 


vanA
M97297
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1141


 


vanA
—
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1051


 


vanA
—
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1052


 


vanA
—
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1053


 


vanA
—
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1054


 


vanA
—
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1055


 


vanA
—
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1056


 


vanA
—
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1057


 


vanA
—
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1049


 


vanA
—
A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA
1050


 


vanB
U94526
C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
1117


 


vanB
U94527
C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2556


 


vanB
U94528
C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2557


 


vanB
U94529
C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2558


 


vanB
U94530
C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2559


 


vanB
Z83305
C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2560


 


vanB
U81452
C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2561


 


vanB
U35369
C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2562


 


vanB
U72704
C TGCGATAGAA GCgGCAGGAC AATAT...ATG GTATCTTCCG CATCCATCAG GAA
−2563


 


vanB
L06138
C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2564


 


vanB
L15304
C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2565


 


vanB
U00456
C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA
−2566


 


vanD
AF130997
C AGCaATcGAA GaAGCAaGAa AATAT...ACG GctTtTTtaa gATtCATCAG GAA
−2567


 


vanE
AF136925
A AGCaATAGAc GaAGCttcAa AATAT...ATG GctTtTTCga CtatgAagAG AAA
−2568


 









Selected sequence
    CGATAGAA GCAGCAGGAC AA
1095


for amplification




primer




 


Selected sequence
                                    GTATCTTCCG CATCCATCAG
1096


for amplification




primera





The sequence numbering refers to the Enterococcus faecium vanB gene fragment (SEQ ID NO. 1117).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequences displayed.


aThis sequence is the reverse-complement of the above vanB sequence.













TABLE 68







Strategy for the selection of vanC-specific


amplification primers from vanC sequences.











Accession #
929                          957   1064                        1092
SEQ ID NO.













vanC1
—
GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT
1058


 


vanC1
—
GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT
1059


 


vanC1
M75132
GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT
1138


 


vanC2
—
GT AGACGGCTTT TTCGATTTTG AAGAGAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
1060


 


vanC2
—
GT AGACGGCTTT TTCGATTTTG AAGAGAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
1061


 


vanC2
—
GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
1062


 


vanC2
—
GT AGACGGCTTT TTCGATTTTG AAGAGAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
1063


 


vanC2
L29638
GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
−2569


 


vanC2
L29638
GT AGACGGCTTT TTCGATTTTG AAGAGAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
−2570


 


vanC3
—
GT AGACGGCTTT TTCGATTTTG AAGAGAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
1064


 


vanC3
—
GT AGACGGCTTT TTCGATTTTG AAGAGAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
1065


 


vanC3
—
GT AGACGGCTTT TTCGATTTTG AAGAGAA...AAAGGaC TTGCTCGCAT CGACTTTTTT GT
1066


 


vanC3
L29639
GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT
−2571


 









Selected sequence
    GACGGYTTT TTYGATTTTG AAGA
1101


for resistance




primer




 


Selected sequence
                                      GGTC TKGCTCGMAT CGAYTTTTT
1102


for resistance




primera





The sequence numbering refers to the vanC1 gene fragment (SEQ ID NO. 1138).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequence displayed.


“R” “Y” “M” “K” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“W” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“D” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C,, G or T.


aThis sequence is the reverse-complement of the selected sequence.













TABLE 69







Strategy for the selection of Streptococcus pneumoniae-specific


amplification primers and hybridization probes from pbp1a sequences.











Accession #
453                                                     505   678
SEQ ID NO.:





pbp1a
M90528
A TTGACTAcCC AAGCATaCAc TATGCtAAtG CtATTTCAAG TAATACAACC GA...TATATG
−2572


 


pbp1a
X67873
A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAACACAACC GA...TATATG
−2573


 


pbp1a
AB006868
A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAACACAACC GA...TATATG
−2574


 


pbp1a
AF046234
A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAACACAACC GA...TATATG
−2575


 


pbp1a

A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAACACAACC GA...TATATG
1014


 


pbp1a

A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAACACAACC GA...TACATG
1017


 


pbp1a
AB006873
A TCGACTAcCC AAGtcTtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG
−2576


 


pbp1a
AF139883
A TCGACTATCC AAGCATGCAT TATCCAAACG CCATTTCAAG TAATACAACA GA...TATATG
1169


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG
1004


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG
1007


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG
1008


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG
1009


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG
1011


 


pbp1a
AF159448
A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG
−2577


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG
1005


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG
1015


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG
1006


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG
1012


 


pbp1a
X67867
A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG
−2578


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG
1010


 


pbp1a
Z49094
A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG
−2579


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG
1013


 


pbp1a

A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG
1016


 


pbp1a
X67870
A TCGACTATCC AAGtATGCAT TAcGCAAACG CCATTTCAAG TAACACAACT GA...TATATG
−2580


 


pbp1a

A TTGACTATCC AAGtATtCAc TActCAAAtG CtATTTCAAG TAATACAACT GA...TATATG
1018


 


pbp1a
AJ002290
A TTGAtTAcCC AActATGgtc TATGCtAACG CtATTTCAAG TAATACAACT GA...TACATG
−2581


 


pbp1a
X67871
A TCGACTAcCC AAGtcTtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TACATG
−2582


 









Selected sequences
    GACTATCC AAGCATGCAT TATG
1130


for amplification
                                                                 ATG



primers




 


Selected sequence
                            CAAACG CCATTTCAAG TAATACAAC
1197


for hybridization




probe
















Accession #
                      706
SEQ ID NO.:






pbp1a
M90528
ATGACaGAtA TGATGAAAAC CGT...
−2572


 



pbp1a
X67873
ATGACCGAAA TGATGAAAAC AGT...
−2573


 



pbp1a
AB006868
ATGACCGACA TGATGAAAAC AGT...
−2574


 



pbp1a
AF046234
ATGACCGAAA TGATGAAAAC TGT...
−2575


 



pbp1a

ATGACCGACA TGATGAAAAC TGT...
1014


 



pbp1a

ATGACCGAAA TGATGAAAAC TGT...
1017


 



pbp1a
AB006873
ATGACCGACA TGATGAAAAC AGT...
−2576


 



pbp1a
AF139883
ATGACCGACA TGATGAAAAC AGT...
1169


 



pbp1a

ATGACCGACA TGATGAAAAC AGT...
1004


 



pbp1a

ATGACCGACA TGATGAAAAC AGT...
1007


 



pbp1a

ATGACCGACA TGATGAAAAC AGT...
1008


 



pbp1a

ATGACCGACA TGATGAAAAC AGT...
1009


 



pbp1a

ATGACCGACA TGATGAAAAC AGT...
1011


 



pbp1a
AF159448
ATGACCGACA TGATGAAAAC AGT...
−2577


 



pbp1a

ATGACCGAAA TGATGAAAAC TGT...
1005


 



pbp1a

ATGACCGAAA TGATGAAAAC TGT...
1015


 



pbp1a

ATGACCGAAA TGATGAAAAC TGT...
1006


 



pbp1a

ATGACCGAAA TGATGAAAAC TGT...
1012


 



pbp1a
X67867
ATGACCGAAA TGATGAAAAC TGT...
−2578


 



pbp1a

ATGACtGAAA TGATGAAAAC TGT...
1010


 



pbp1a
Z49094
ATGACtGAAA TGATGAAAAC TGT...
−2579


 



pbp1a

ATGACtGAAA TGATGAAAAC TGT...
1013


 



pbp1a

ATGACtGAAA TGATGAAAAC TGT...
1016


 



pbp1a
X67870
ATGACCGAAA TGATGAAAAC TGT...
−2580


 



pbp1a

ATGACtGAAA TGATGAAAAC TGT...
1018


 



pbp1a
AJ002290
ATGACtGAAA TGATGAAAAC AGT...
−2581


 



pbp1a
X67871
ATGACaGAAA TGATGAAAAC AGT...
−2582


 











Selected sequences





for amplification
ATGACHGAMA TGATGAAAAC
1129



primers




 



Selected sequence





for hybridization





probe









The sequence numbering refers to the Streptococcus pneumoniae pbp1a gene fragment (SEQ ID NO. 1004).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lower-case letters.


Dotes indicate gaps in the sequences displayed.


“R” “Y” “N” “K” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“N” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“H” stands for A, C or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four


nucleotides A, C, G or T.














Accession #
   756                        783   813                         840
SEQ ID NO.:





pbp1a
M90528
...GCTGGTAA aACtCGTACg TCaAACTATA...A ATACgGGTTA TGTAGCTCCG GAcGAAA
−2583


 


pbp1a
X67873
...GCTGGTAA aACAGGaACc TCTAACTATA...A CCtCTcaaTt TGTACCaCCt GATCAAC
−2584


 


pbp1a
AB006868
...GCTGGTAA aACACGaACc TCTAACTATA...A CCtCTcaaTt TCTAGCaCCt CAcCAAC
−2585


 


pbp1a
AF046234
...GCAGGTAA aACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA
−2586


 


pbp1a

...GCACGTAA aACAGCTACT TCTAACTATA...A ACACTGGTTA CCTACCTCCA GATGAAA
1014


 


pbp1a

...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1017


 


pbp1a
AB006873
...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA
−2587


 


pbp1a
AF139883
...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTCGCTA TGTAGCTCCA GATGAAA
1169


 


pbp1a

...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1004


 


pbp1a

...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1007


 


pbp1a

...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1008


 


pbp1a

...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1009


 


pbp1a

...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1011


 


pbp1a
AF159448
...GCTGGTAA aACAGGaACC TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA
−2588


 


pbp1a

...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1005


 


pbp1a

...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1015


 


pbp1a

...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1006


 


pbp1a

...GCTGGTAA GACACGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA
1012


 


pbp1a
X67867
...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA
−2589


 


pbp1a

...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA
1010


 


pbp1a
Z49094
...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA
−2590


 


pbp1a

...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA
1013


 


pbp1a

...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA
1016


 


pbp1a
X67870
...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA
−2591


 


pbp1a

...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA
1018


 


pbp1a
AJ002290
...GCAGGTAA GACgGGTACa TCTAACTACA...A ACACTGGCTA C~~~~~~~~~ ~~~~~~~
−2592


 


pbp1a
X67871
...GCTGGTAA aACAGGTACc TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA
−2593


 









Selected sequence
      GGTAA GACAGGTACT TCTAACT
1193


for hybridization




probe




Selected sequence
                                        ACTGGYTA YGTAGCTCCA GATG
1131


for amplification




primera





The sequence numbering refers to the Streptococcus pneumoniae pbp1a gene fragment (SEQ ID NO. 1004).


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lower case letters.


Dots indicate gaps in the sequences displayed.


“~” indicates incomplete sequence data.


“R” “Y” “W” “and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.


This sequence is the reverse-complement of the selected primer.













TABLE 70







Specific and ubiquitous primers for nucleic


acid amplification (toxin sequences).











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Toxin gene:
cdtA




2123 
5′-TCT ACC ACT GAA GCA TTA C
2129a
442-460


2124b
5′-TAG GTA CTG TAG GTT TAT TG
2129a
580-599


 


Toxin gene:
cdtB




2126 
5′-ATA TCA GAG ACT GAT GAG
2130a
2665-2682


2127b
5′-TAG CAT ATT CAG AGA ATA TTG T
2130a
2746-2767


 


Toxin gene:
stx1




1081 
5′-ATG TCA GAG GGA TAG ATC CA
1076a
233-252


1080b
5′-TAT AGC TAC TGT CAC CAG ACA ATG T
1076a
394-418


 


Toxin gene:
stx2




1078 
5′-AGT TCT GCG TTT TGT CACTGT C
1077a
546-567


1079b
5′-CGG AAG CAC ATT GCT GAT T
1077a
687-705


 


Toxin genes:
stx1 and stx2




1082 
5′-TTG ARC RAA ATA ATT TAT ATG TG
1076a
278-300


1083b
5′-TGA TGA TGR CAA TTC AGT AT
1076a
781-800





aSequences from databases.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.













TABLE 71







Molecular beacon internal hybridization probes


for specific detection of toxin sequences.











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequencea
SEQ ID NO.
position





Toxin gene:
cdtA




2125b
5′-CAC GCG GAT TTT GAA TCT CTT CCT CTA
2129c
462-488



GTA GCG CGT G




 


Toxin gene:
cdtB




2128 
5′-CAA CGC TGG AGA ATC TAT ATT TGT AGA
2130c
2714-2740



AAC TGC GTT G




 


Toxin gene:
stx1




1084 
5′-CCA CGC CGC TTT GCT GAT TTT TCA CAT
1076c
337-363



GTT ACC GCG TGG




 


2012d
5′-CCG CGG ATT ATT AAA CCG CCC TTC CGC
1076c
248-264



GG-MR-HEG-ATG TCA GAG GGA TAG ATC CA




 


Toxin gene:
stx2




1085 
5′-CCA CGC CAC TGT CTG AAA CTG CTC CTG
1077c
617-638



TG CGT GG





aUnderlined nucleotides indicate the molecular beacon's stem.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.


cSequences from databases.


dScorpion primer.













TABLE 72







Specific and ubiquitous primers for


nucleic acid amplification (van sequences).











Originating DNA fragment












SEQ ID
Nucleotide


SEQ ID NO.
Nucleotide sequence
NO.
position













Resistance gene:
vanA




1086 
5′-CTA CTC CCG CCT TTT GGG TT
1049-1057a
 513-532b


1087c
5′-CTC ACA GCC CGA AAC AGC CT
1049-1057a
 699-718b


 


1086 
5′-CTA CTC CCG CCT TTT GGG TT
1049-1057a
 513-532b


1088c
5′-TGC CGT TTC CTG TAT CCG TC
1049-1057a
 885-904b


 


1086 
5′-CTA CTC CCG CCT TTT GGG TT
1049-1057a
 513-532b


1089c
5′-ATC CAC ACG GOC TAG ACC TC
1049-1057a
 933-952b


 


1090 
5′-AAT AGC GCG GAC GAA TTG GAC
1049-1057a
 629-649b


1091c
5′-AAC GCG GCA CTG TTT CCC AA
1049-1057a
 734-753b


 


1090 
5′-AAT AGC GCG GAC GAA TTG GAC
1049-1057a
 629-649b


1089c
5′-ATC CAC ACG GGC TAG ACC TC
1049-1057a
 933-952b


 


1092 
5′-TCG GCA AGA CAA TAT GAC AGC
1049-1057a
 662-682b


1088c
5′-TGC CGT TTC CTG TAT CCG TC
1049-1057a
 885-904b


 


Resistance gene:
van.B




1095 
5′-CGA TAG AAG CAG CAG GAC AA
1117d
473-492


1096c
5′-CTG ATG GAT GCG GAA GAT AC
1117d
611-630


 


Resistance genes:





1112 
5′-GGC TGY GAT ATT CAA AGC TC
1049-1057, 1117a
 437-456b


1113c
5′-ACC GAC CTC ACA GCC CGA AA
1049-1057, 1117a
 705-724b


 


1112 
5′-GGC TGY GAT ATT CAA AGC TC
1049-1057, 1117a
 437-456b


1114c
5′-TCW GAG CCT TTT TCC GGC TCG
1049-1057, 1117a
 817-837b


 


1115 
5′-TTT CGG GCT GTG AGG TCG GBT GHG CG
1049-1057, 1117a
 705-730b


1114c
5′-TCW GAG CCT TTT TCC GGC TCG
1049-1057, 1117a
 817-837b


 


1116 
5′-TTT CGG GCT GTG AGG TCG GBT GHG CGG
1049-1057, 1117a
 705-731b


1114c
5′-TCW GAG CCT TTT TCC GGC TCG
1049-1057, 1117a
 817-837b


 


1112 
5′-GGC TGY GAT ATT CAA AGC TC
1049-1057, 1117a
 437-456b


1118c
5′-TTT TCW GAG CCT TTT TCC GGC TCG
1049-1057, 1117a
 817-840b







a These sequences were aligned to derive the corresponding primer.


b The nucleotide positions refer to the vanA sequence fragment


(SEQ ID NO. 1051)


c These sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


d Sequences from databases.













Resistance genes:
vanA, vanE




1115 
5′-TTT CGG GCT GTG AGG TCG GBT GHG CG
1049-1057, 1117a
705-730b


1118c
5′-TTT TCW GAG CCT TTT TCC GGC TCG
1049-1057, 1117a
817-840b


 


1116 
5′-TTT CGG GCT GTG AGG TCG GBT GHG CGG
1049-1057, 1117a
705-731b


1118c
5′-TTT TCW GAG CCT TTT TCC GGC TCG
1049-1057, 1117a
817-840b


 


1119 
5′-TTT CGG GCT GTG AGG TCG GBT GHG C
1049-1057, 1117a
705-729b


1118c
5′-TTT TCW GAG CCT TTT TCC GGC TCG
1049-1057, 1117a
817-840b


 


1120 
5′-TTT CGG GCT GTG AGG TCG GBT GHG
1049-1057, 1117a
705-728b


1111c
5′-TTT TCW GAG CCT TTT TCC GGC TCG
1049-1057, 1117a
817-840b


 


1121 
5′-TGT TTG WAT TGT CYG GYA TCC C
1049-1057, 1117a
408-429b


1111c
5′-CTT TTT CCG GCT CGW YTT CCT GAT G
1049-1057, 1117a
806-830b


 


1112 
5′-GGC TGY GAT ATT CAA AGC TC
1049-1057, 1117a
437-456b


1111c
5′-CTT TTT CCG GCT CGW YTT CCT GAT G
1049-1057, 1117a
806-830b


 


1123 
5′-TTT CGG GCT GTG AGG TCG GBT G
1049-1057, 1117a
705-726b


1111c
5′-CTT TTT CCG GCT CGW YTT CCT GAT G
1049-1057, 1117a
806-830b


 


1112 
5′-GGC TGY GAT ATT CAA AGC TC
1049-1057, 1117a
437-456b


1124c
5′-GAT TTG RTC CAC YTC GCC RAC A
1049-1057, 1117a
757-778b


 


Resistance gene:
vanC1




1103 
5′-ATC CCG CTA TGA AAA CGA TC
1058-1059a
519-538d


1104c
5′-GGA TCA ACA CAG TAG AAC CG
1058-1059a
678-697d


 


Resistance genes:
vanC1, vanC2, vanC3




1097 
5′-TCY TCA AAA GGG ATC ACW AAA GTM AC
1058-1066a
607-632d


1098c
5′-TCT TCA AAA TCG AAA AAG CCG TC
1058-1066a
787-809d


 


1099 
5′-TCA AAA GGG ATC ACW AAA GTM AC
1058-1066a
610-632d


1100c
5′-GTA AAK CCC GGC ATR GTR TTG ATT TC
1058-1066a
 976-1001d


 


1101 
5′-GAC GGY TTT TTY GAT TTT GAA GA
1058-1066a
787-809d


1102c
5′-AAA AAR TCG ATK CGA GCM AGA CC
1058-1066a
922-944d


 


Resistance genes:
vanC2, vanC3




1105 
5′-CTC CTA CGA TTC TCT TGA YAA ATC A
1060-1066, 1140a
487-511e


1106c
5′-CAA CCG ATC TCA ACA CCG GCA AT
1060-1066, 1140a
690-712e







a These sequences were aligned to derive the corresponding primer.


b The nucleotide positions refer to the vanA sequence fragment


(SEQ ID NO. 1051)


c These sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


d The nucleotide positions refer to the vanC1 sequence fragment


(SEQ ID NO. 1058)


e The nucleotide positions refer to the vanc2 sequence fragment


(SEQ ID NO. 1140)













Resistance gene:
vanD




1591 
5′-ATG AGG TAA TAG AAC GGA TT
1594
797-837


1592b
5′-CAG TAT TTC AGT AAG CGT AAA
1594
979-999


 


Resistance gene:
vanE




1595 
5′-AAA TAA TGC TCC ATC AAT TTG CTG A
1599a
74-98


1596b
5′-ATA GTC GAA AAA GCC ATC CAC AAG
1599a
394-417


 


1597 
5′-GAT GAA TTT GCG AAA ATA CAT GGA
1599a
163-186


1598b
5′-CAG CCA ATT TCT ACC CCT TTC AC
1599a
319-341


 



Sequencing primers (vanA.B)




1112 
5′-GGC TGY GAT ATT CAA AGC TC
1139a
737-756


1111b
5′-CTT TTT CCG GCT CGW YTT CCT GAT G
1139a
1106-1130


 



Sequencing primers (vanA, vanX, vanY)




1150 
5′-TGA TAA TCA CAC CGC ATA CG
1141a
860-879


1151b
5′-TGC TGT CAT ATT GTC TTG CC
1141a
1549-1568


 


1152 
5′-ATA AAG ATG ATA GGC CGG TG
1141a
1422-1441


1153b
5′-CTC GTA TGT CCC TAC AAT GC
1141a
2114-2133


 


1154 
5′-GTT TGA AGC ATA TAG CCT CG
1141a
2520-2539


1155b
5′-CAG TGC TTC ATT AAC GTA GTC
1141a
3089-3109







a Sequences from databases.


b These sequences are from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.














Sequencing primers (vanC1)




1110 
5′-ACG AGA AAG ACA ACA GGA AGA CC
1138a
122-144


1109b
5′-ACA TCG TGA TCG CTA AAA GGA GC
1138a
1315-1337


 



Sequencing primers (vanC2, vanC3)




1108 
5′-GTA AGA ATC GGA AAA GCG GAA GG
1140a
 1-23


1107b
5′-CTC ATT TGA CTT CCT CCT TTG CT
1140a
1064-1086





aSequences from databases.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.













TABLE 73







Internal hybridization probes for


specific detection of van sequences.












Originating DNA fragment














Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Resistance gene:
vanA




1170
5′-ACG AAT TGG ACT ACG CAA TT
1049-1057a
 639-658b


 


2292
5′-GAA TCG GCA AGA CAA TAT G
2293c
583-601


 


Resistance gene:
vanE




1171
5′-ACG AGG ATG ATT TGA TTG TC
1117c
560-579


 


2294
5′-AAA CGA GGA TGA TTT GAT TG
2296a
660-679


 


2295
5′-TTG AGC AAG CGA TTT CGG
2296a
614-631


 


Resistance gene:
vanD




2297
5′-TTC AGG AGG GGG ATC GC
1594c
458-474





aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the vanA sequence fragment (SEQ ID NO. 1051)


cSequences from databases.













TABLE 74







Specific and ubiquitous primers for


nucleic acid amplification (pbp sequences).











Originating DNA fragment










SEQ ID NO.
Nucleotide sequence
NO.
position













Resistance gene:
pbp1a




1129 
5′-ATG ATG ACH GAM ATG ATG AAA AC
1004-1018a
 681-703b


1131c
5′-CAT CTG GAG CTA CRT ARC CAG T
1004-1018a
 816-837b


 


1130 
5′-GAC TAT CCA AGC ATG CAT TAT G
1004-1018a
 456-477b


1131 
5′-CAT CTG GAG CTA CRT ARC CAG T
1004-1018a
 816-837b


 


2015 
5′-CCA AGA AGC TCA AAA ACA TCT G
2047d
909-930


2O16c
5′-TAD CCT GTC CAW ACA GCC AT
2047d
1777-1796


 



Sequencing primers (pbp1a)




1125 
5′-ACT CAC AAC TGG GAT GGA TG
1169d
873-892


1126c
5′-TTA TGG TTG TGC TGG TTG AGG
1169d
2140-2160


 


1125 
5′-ACT CAC AAC TGG GAT GGA TG
1169d
873-892


1128c
5′-GAC GAC YTT ATK GAT ATA CA
1169d
1499-1518


 


1127 
5′-KCA AAY GCC ATT TCA AGT AA
1169d
1384-1403


1126c
5′-TTA TGG TTG TGC TGG TTG AGG
1169d
2140-2160


 



Sequencing primers (pbp2b)




1142 
5′-GAT CCT CTA ART GAT TCT CAG GTG G
1172d
 1-25


1143c
5′-CAR TTA GCT TAG CAR TAG GTG TTG G
1172d
1481-1505


 


1142 
5′-GAT CCT CTA ART GAT TCT CAG GTG G
1172d
 1-25


1145c
5′-ARC ATA TTK GGT TGA TAG GT
1172d
793-812


 


1144 
5′-TGT YTT CCA AGG TTC AGC TC
1172d
657-676


1143c
5′-CAR TTA GCT TAG CAA TAG GTG TTG G
1172d
1481-1505


 



Sequencing primers (pbp2x)




1146 
5′-GGG ATT ACC TAT GCC ART ATG AT
1173d
219-241


1147c
5′-AGC TGT GTT AGC VCG ARC ATC TTG
1173d
1938-1961


 


1146 
5′-GGG ATT ACC TAT GCC ART ATG AT
1173d
219-241


1149c
5′-TCC YAC WAT TTC TTT TTG WG
1173d
1231-1250


 


1148 
5′-GAC TTT GTT TGG CGT GAT AT
1173d
711-730


1147c
5′-AGC TGT GTT AGC VCG ARC ATC TTG
1173d
1938-1961





aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the pbp1a sequence fragment (SEQ ID NO. 1004)


cThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.


dSequences from databases.













TABLE 75







Internal hybridization probes for


specific detection of pbp sequences.











Originating DNA fragment












SEQ ID
Nucleotide


SEQ ID NO.
Nucleotide sequence
NO.
position





Resistance gene:
pbp1a




1132
5′-AGT GAA AAR ATG GCT GCT GC
1004-1018a
 531-550b


 


1133
5′-CAT CAA GAA CAC TGG CTA YGT AG
1004-1018a
 806-828b


 


1134
5′-CTA GAT AGA GCT AAA ACC TTC CT
1004-1018a
 417-439b


 


1135
5′-CAT TAT GCA AAC GCC ATT TCA AG
1004-1018a
 471-493b


 


1192
5′-GGT AAA ACA GGA ACC TCT AAC T
1004-1018a
 759-780b


 


1193
5′-GGT AAG ACA GGT ACT TCT AAC T
1004-1018a
 759-780b


 


1194
5′-CAT TTC AAG TAA TAC AAC AGA ATC
1004-1018a
 485-508b


 


1195
5′-CAT TTC AAG TAA CAC AAC TGA ATC
1004-1018a
 485-508b


 


1196
5′-GCC ATT TCA AGT AAT ACA ACA GAA
1004-1018a
 483-506b


 


1197
5′-CAA ACG CCA TTT CAA GTA ATA CAA C
1004-1018a
 478-502b


 


1094
5′-GGT AAA ACA GGT ACT TCT AAC TA
1004-1018a
 759-781b


 


1214
5′-GGT AAA ACA GGT ACC TCT AAC TA
1004-1018a
 759-781b


 


1216
5′-GGT AAG ACT GGT ACA TCA AAC TA
1004-1018a
 759-781b


 


1217
5′-CAA ATG CCA TTT CAA GTA ACA CAA C
1004-1018a
 478-502b


 


1218
5′-CAA ACG CCA TTT CAA GTA ACA CAA C
1004-1018a
 478-502b


 


1219
5′-CAA ATG CTA TTT CAA GTA ATA CAA C
1004-1018a
 478-502b


 


1220
5′-CAA ACG CCA TTT CAA GTA ATA CGA C
1004-1018a
 478-502b


 


2017
5′-ACT TTG AAT AAG GTC GGT CTA G
2047c
1306-1327


 


2018
5′-ACA CTA AAC AAG GTT GGT TTA G
2063 
354-375


 


2019
5′-ACA CTA AAC AAG GTC GGT CTA G
2064 
346-367


 


2020
5′-GTA GCT CCA GAT GAA ATG TTT G
2140c
1732-1753


 


2021
5′-GTA GCT CCA GAC GAA ATG TTT G
2057 
831-852


 


2022
5′-GTA GCT CCA GAT GAA ACG TTT G
2053c
805-826


 


2023
5′-GTA ACT CCA GAT GAA ATG TTT G
2056 
819-840


 


2024
5′-AGT GAA AAG ATG GCT GCT GC
2048c
1438-1457


 


2025
5′-AGT GAG AAA ATG GCT GCT GC
2047c
1438-1457


 


2026
5′-TCC AAG CAT GCA TTA TGC AAA CG
2047c
1368-1390


 


2027
5′-TCG GTC TAG ATA GAG CTAAAA CG
2047c
1319-1341


 


2028
5′-TAT GCT CTT CAA CAA TCA CG
2047c
1267-1286


 


2029
5′-AGC CGT TGA GAC TTT GAA TAA G
2047c
1296-1317


 


2030
5′-CTT AAT GGT CTT GGT ATC G
2047c
1345-1366


 


2031
5′-CGT GAC TGG GGT TCT GCT ATG A
2049c
1096-1117


 


2032
5′-CGT GAC TGG GGA TCA TCA ATG A
2047c
1096-1117


 


2033
5′-CGT GAC TGG GGT TCT GCC ATG A
2057 
195-216


 


2034
5′-ATC AAG AAC ACT GGC TAT GTA G
2050c
787-808


 


2035
5′-ATC AAG AAC ACT GGC TAC GTA G
2051c
787-808


 


2036
5′-ATC AAG AAC ACT GGT TAC GTA G
2047 
1714-1735


 


2037
5′-ATC AAA AAT ACT GGT TAT GTA G
2057 
813-834


 


2038
5′-ATC AAG AAT ACT GGC TAC GTA G
2052c
757-778


 


2039
5′-ATC AAA AAC ACT GGC TAT GTA G
2053c
787-808





aThese sequences were aligned to derive the corresponding primer.


bThe nucleotide positions refer to the pbpla sequence fragment (SEQ ID NO. 1004)


cSequence from databases? 













TABLE 76







Strategy for the selection of vanAB-specific amplification primers


and vanA- and vanB- specific hybridization probes from van sequences











Accession #

SEQ ID NO.:















734                      759       936                      961



vanA
X56895
GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1139


 


vanA
M97297
GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1141


 


vanA

GTAGCCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1051


 


vanA

GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACCAATT GGACTACGCA ATTGAA. . .
1052


 


vanA

GTAGGCT CCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1053


 


vanA

GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1054


 


vanA

GTAGGCT CCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1055


 


vanA

GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1056


 


vanA

GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1057


 


vanA

GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1049


 


vanA

GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA. . .
1050


 


vanB
U94526
GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA. . .
1117


 


vanB
U94527
CTAGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT aaACgctGCg ATaGAA. . .
−2594


 


vanB
U94528
GTGCGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA. . .
−2595


 


vanB
U94529
GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA. . .
−2596


 


vanB
U94530
GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA. . .
−2597


 


vanB
Z83305
GTGCGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA. . .
−2598


 


vanB
U81452
GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA. . .
−2599


 


vanB
U35369
GTAGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT aaACgctGCg ATaGAA. . .
−2600


 


vanB
U72704
GTGGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA. . .
−2601


 


vanB
L06138
GTAGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT aaACgctGCg ATaGAA. . .
−2602


 


vanB
L15304
GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA. . .
−2603


 


vanB
U00456
GTAGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT aaACgctGCg ATaGAA. . .
−2604


 


vanD
AF130997
GTGGGaT GCGATATTCA AAGCTCCGT . . . CAGAaGAAcT GcAggcaGCA ATcGAA. . .
−2605


 


vanE
AF136925
GTAGGtT GTGgTATcgg AgctgCAGC . . . AAAgtGAtTT atAtaAaGCA ATaGAC. . .
−2606


 









Selected sequence for
   GGCT GYGATATTCA AA-CTC
1112


amplification primer




 


Selected sequence for
                                       ACGAATT GGACTACGCA ATT (vanA)
1170


hybridization probe




 







The sequence numbering refers to the Enterococcus faecium vanA gene fragment (SEQ ID NO. 1139)


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lower-case letters.


Dots indicate gaps in the sequences displayed.


“R” “Y” “M” “K” “W” and “S” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“Y” stands for C or T;


“M” stands for A or C;


“K” stands for G or T;


“W” stands for A or T;


“S” stands for C or G.


“I” stands for inosine which is a nucleotide analog that can bind to any of the four


nucleotides A, C, G or T.















1038                    1063       1103                          1133



vanA
X56895
GAAACagt GccGcGTTag TTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1139


 


vanA
M97297
GAAACagt GccGcgTTag TTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1141


 


vanA

GAAACagt GccGcgTTag TTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1051


 


vanA

GAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1052


 


vanA

GAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1053


 


vanA

GAAACagt GccGcgTTag TTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1054


 


vanA

GAAACagt GCCGCgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1055


 


vanA

GAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1056


 


vanA

GAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1057


 


vanA

GAAACagt GccGcgTTag CTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1049


 


vanA

GAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT
1050


 


vanB
U94526
GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
1117


 


vanB
U94527
GAAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2607


 


vanB
U94528
GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2608


 


vanB
U94529
GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2609


 


vanB
U94530
GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2610


 


vanB
Z83305
GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2611


 


vanB
U81452
GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2612


 


vanB
U35369
GAAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2613


 


vanB
U72704
GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGAT
−2614


 


vanB
L06138
GAAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2615


 


vanB
L15304
GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2616


 


vanB
U00456
GAAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT
−2617


 


vanD
AF130997
GAAACGga aATGATcTCA TgGctGGC . . . ATT CATCAGGAAG cacAGCCGGA aAAGGGAT
−2618


 


vanE
AF136925
GAAA...t GAacAaTTGg TcGTtGGA . . . TAT gAagAGaAAt ACaA...... ......TT
−2619


 









Selected sequence for
   ACGAG GATGATTTGA TTGTC (vanE)
1171


hybridization probe




 


Selected sequence for
                                       CATCAGGAAR WCGAGCCGGA AAAAG
1111


amplification primera





The sequence numbering refers to the Enterococcus faecium vanA gene fragment (SEQ ID NO. 1139)


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lowercase letters.


Dots indicate gaps in the sequences displayed


“R” and “W” designate nucleotide positions which are degenerated.


“R” stands for A or G;


“W” stands for A or T


aThis sequence is the reverse-complement of the above selected primer.













TABLE 77







Internal hybridization probe for specific detection of mecA.











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Resistance gene:
mecA




1177
5′-GCT CAA CAA GTT CCA GAT TA
1178a
1313-1332





aSequence from databases.













TABLE 78







Specific and ubiquitous primers for nucleic


acid amplification (hexA sequences).











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position













Bacterial species:
Streptococcus pneumoniae




1179 
5′-ATT TGG TGA CGG GTG ACT TT
1183a
431-450 


1181b
5′-AGC AGC TTA CTA GAT GCC GT
1831-1191c
652-671d


 



Sequencing primers




1179 
5′-ATT TGG TGA CGG GTG ACT TT
1183a
431-450


1182b
5′-AAC TGC AAG AGA TCC TTT GG
1183a
1045-1064





aSequences from databases.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.


cThese sequences were aligned to derive the corresponding primer.


dThe nucleotide positions refer to the hexA sequence fragment (SEQ ID NO. 1183)













TABLE 79







Internal hybridization probe for


specific detection of hexA sequences.











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Bacterial species:
Streptococcus pneumoniae




1180a
5′-TCC ACC GTT GCC AAT CGC A
1183-1191b
629-647c





aThis sequences is from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.


bThese sequences were aligned to derive the corresponding primer.


cThe nucleotide positions refer to the hexA sequence fragment (SEQ ID NO. 1183)













TABLE 80







Strategy for the selection of Streptococcus pneumoniae species-specific


amplification primers and hybridization probe from hexA sequences.











SEQ




ID




NO.






428                       453       626



S. pneumoniae
TGG ATTTGGTGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGAGCA AACGGCATCT AGTAAGCTGC
1183


 


S. pneumoniae
~~~~~~~~~TGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGACCA AACGGCATCT AGTAAGCTGC
1184


 


S. pneumoniae
~~~~~~~~~TGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGAGCA AACGGCATCT AGTAAGCTGC
1185


 


S. pneumoniae
~~~~~~~~~TGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGAGCA AACGGCATCT AGTAAGCTGC
1186


 


S. pneumoniae
~~~~~~~~~TGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGAGCA AACGGCATCT AGTAAGCTGC
1187


 


S. oralis
~~~~~~~~~~~~~GGGTGACTTT TAT . . . ATCca CGACTGGCAg CtGTCGAGCA AgCGGCAgCT AGTAAGCTcC
1188


 


S. mitis
~~~~~~~GGTGAC GGGTGACTTT TAT . . . ATTca CGATTGGCAg CtGTGGAGCA AgcGGCATCT AGTAAaCTGC
1189


 


S. mitis
~~~~~~~~~TGAC GGGTGACTTT CAG . . . GCGaG gagcTGtCtc CtaTGGAGCG TcaGGCAgca gGgAAaCTGc
1190


 


S. mitis
~~~~~~~~~TGAC GGG-GACTTT CAG . . . GCGAG gaAcTGtCtc CtaTGGAGCG TcaGGCAgcg gGgAAatTGC
1191


 


Selected
    ATTTGGTGAC GGGTGACTTT
1179


sequence for




amplification




primer




 


Selected
                                                                 ACGGCATCT AGTAAGCTGC



sequences for




amplification




primersa




Selected
                                       TG CGATTGGCAA CCGTGGA
1180


sequence for




hybridization




probea




 












674        1042                    1067




S. pneumoniae
TCCA . . . AATCCAAAG GATCTCTTGC AGTTGGC
1183


 



S. pneumoniae
TCCA . . . AATCCAAAG GATCTCTTG~~~~~~~~
1184


 



S. pneumoniae
TCCA . . . AATCCAAAG GATCTC~~~~~~~~~~~
1185


 



S. pneumoniae
TCCA . . . AATCCAAAG GATCTCTT~~~~~~~~~
1186


 



S. pneumoniae
TCCG . . . ATCCAAAAG GATCTCTT~~~~~~~~~
1187


 



S. oralis
TCCA . . . ~~~~~~~~~~~~~~~~~~~~~~~~~~
1188


 



S. mitis
TTCA . . . AATCCAAAG GATCTCTT~~~~~~~~~
1189


 



S. mitis
TGGA . . . ~~~~~~~~~~~~~~~~~~~~~~~~~~
1190


 



S. mitis
TAGA . . . AATCCAAAG GATCTCTT~~~~~~~~~
1191


 



Selected
T
1181



sequences for
              CCAAAG GATCTCTTGC AGTT
1182



amplification





primersa





The sequence numbering refers to the Streptococcus pneumoniae hexA gene fragment (SEQ ID NO. 1183)


Nucleotides in capitals are identical to the selected sequences or match those sequences.


Mismatches are indicated by lower-case letters.


Dots indicate gaps in the sequences displayed.


“~” indicate in complete sequence data.


aThis sequence is the reverse-complement of the selected primer.













TABLE 81







Specific and ubiquitous primers for


nucleic acid amplification (pcp sequence)











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Bacterial species:
Streptococcus pyogenes




1211 
5′-ATT CTT GTA ACA GGC TTT GAT CCC
1215a
291-314


 


1210b
5′-ACC AGC TTG CCC AAT ACA AAG G
1215a
473-494





aSequences from databases.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.













TABLE 82







Specific and ubiquitous primers for nucleic acid amplification


of S. saprophyticus sequences of unknown coding potential.












SEQ ID
Nucleotide


SEQ ID NO.
Nucleotide sequence
NO.
position





Bacterial species:
Staphylococcus saprophyticus




1208 
5′-TCA AAA AGT TTT CTA AAA AAT TTA C
74, 1093, 1198b
169-193c


 


1209a
5′-ACG GGC GTC CAC AAA ATC AAT AGG A
74, 1093, 1198b
355-379c





aThis sequence is from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.


bThese sequences were aligned to derive the corresponding primer.


cThe nucleotide positions refer to the S. saprophyticus unknown gene sequence fragment (SEQ ID NO. 1198).













TABLE 83







Molecular beacon internal hybridization probes for specific


detection of antimicrobial agents resistance gene sequences.











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequencea
SEQ ID NO.
position













Resistance gene:
gyrA




2250 
5′-CCG TCG GAT GGT GTC GTA TAC CGC GGA GTC
1954b
218-243



GCC GAC GG




2251 
5′-CGG AGC CGT TCT CGC TGC GTT ACA TGC TGG
1954b
259-286



TGG CTC CG




 


Resistance gene:
mecA




1231 
5′-GCG AGC CCG AAG ATA AAA AAG AAC CTC TGC
1178b
1291-1315



TGC TCG C




 


Resistance gene:
parC




1938b
5′-CCG CGC ACC ATT GCT TCG TAC ACT GAG GAG
1321c
232-260



TCT CCG CGC GG




 


1939 
5′-CGA CCC GGA TGG TAG TAT CGA TAA TGA TCC
1321c
317-346



GCC AGC GGC CGG GTC G




 


1955b
5′-CGC GCA ACC ATT GCT TCG TAC ACT GAG GAG
1321c
235-260



TCT GCG CG




 


Resistance gene:
vanA




1239 
5′-GCG AGC GCA GAC CTT TCA GCA GAG GAG GCT
1051
860-880



CGC




 


1240 
5′-GCG AGC CGG CAA GAC AAT ATG ACA GCA AAA
1051
663-688



TCG CTC GC




 


Resistance gene:
vanE




1241 
5′-GCG AGC GGG GAA CGA GGA TGA TTT GAT TGG
1117
555-577



CTC GC




 


Resistance gene:
vanD




1593 
5′-CCG AGC GAT TTA CCG GAT ACT TGG CTG ICG
1594
835-845



CTC GG





aUnderlined nucleotides indicate the molecular beacon's stem.


bThis sequence is from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.


cSequence from databases.













TABLE 84







Molecular beacon internal hybridization probe for specific


detection of S. aureus gene sequences of unknown coding potential.











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequencea
SEQ ID NO.
position





Bacterial species:
S. aureus




1232
5′-GGA GCC GCG CGA TTT TAT AAA TGA ATG TTG
1244
53-80



ATA ACC GGC TCC





aUnderlined nucleotides indicate the molecular beacon's stem.













TABLE 85







Molecular beacon internal hybridization probes


for specific detection of tuf sequences.











Originating DNA fragment












SEQ ID
Nucleotide


SEQ ID NO.
Nucleotide sequencea
NO.
position













Bacterial species:
Chlamydia pneumoniae




2091 
5′-CGC GAC TTG AGA TGG AAC TTA GTG AGC
20
157-183



TTC TTG GTC GCG




 


2092 
5′-CGC GAC GAA AGA ACT TCC TGA AGG TCG
20
491-516



TGC AGG TCC AG




 


Bacterial species:
Chlamydia trachomatis




2213 
5′-CGT GCC ATT GAC ATG ATT TCC GAA GAA
1739b
412-441



GAC GCT GAA GGC ACG




 


Bacterial species:
Enterococcus faecalis




1236 
5′-GCG AGC CGT GGT GAA GTT CGC GTT GGT
883
370-391



GGC TCG C




 


Bacterial species:
Enterococcus faecium




1235 
5′-GCG AGC CGA AGT TGA AGT TGT TGG TAT
64
412-437



TGC TGG CTC GC




 


Bacterial species:
Legionella pneumophila




2084c
5′-CAC GCG TCA ACA CCC GTA CAA GTC GTC
112
461-486



TTT TGC GCG TG




 


Bacterial species:
Mycoplasma pneumoniae




2096c
5′-CGC GAC CGG TAC CAC GGC CAG TAA TCG
2097b
658-679



TGT CGC G




 


Bacterial species:
Neisseria gonorrhoeae




2177 
5′-GGC ACG GAC AAA CCA TTC CTG CTG CCT
126
323-357



ATC GAA ACG TGT TCC CGT GCC




 


2178 
5′-GGC ACG ACA AAC CAT TCC TGC TGC CTA
126
323-348



TCG AAC GTG CC




 


2179 
5′-GGC AGC TCT ACT TCC GTA CCA CTG ACG
126
692-718



TAA CCG GCT GCC









a Underlined nucleotides indicate the molecular beacon's stem.


b Sequence from databases.


c This sequence is from the complementary DNA strand of the sequence of the originating


fragment given in the Sequence Listing.













Bacterial species:
Pseudomonas aeruginosa




2122 
5′-CCG AGC GAA TGT AGG AGT CCA GGG TCT
153, 880, 2138b,c
 280-302d



CTG CTC GG




 


Bacterial species:
Staphylococcus aureus




2186 
5′-ACG CGC TCA AAG CAG AAG TAT ACG TAT
1728
615-646



TAT CAA AAG ACG CGC GT




 


Bacterial group:
Staphylococcus sp. other than S. aureus




1233 
5′-GCG AGC GTT ACT GGT GTA GAA ATG TTC
878
372-394



CGG CTC GC




 


Fungal species:
Candida albicans




2073 
5′-CCG AGC AAC ATG ATT GAA CCA TCC ACC
408
404-429



AAC TGG CTC GG




 


Fungal species:
Candida dubliniensis




2074 
5′-CCG AGC AAC ATG ATT GAA GCT TCC ACC
414
416-441



AAC TGG CTC GG




 


Fungal species:
Candida glabrata




2110b
5′-GCG GGC CCT TAA CGA TTT CAG CGA ATC
417
307-335



TGG ATT CAG CCC GC




 


2111 
5′-GCG GGC ATG TTG AAG CCA CCA CCA ACG
417
419-447



CTT CCT GGC CCG C




 


Fungal species:
Candida krusei




2112b
5′-GCG GGC TTG ATG AAG TTT GGG TTT CCT
422
318-347



TGA CAA TTG CCC GC




 


2113 
5′-GCG GGC ACA AGG GTT GGA CTA AGG AAA
422
419-447



CCA AGG CAG CCC GC




 


2114 
5′-GCG GGC ATC GAT GCT ATT GAA CCA CCT
422
505-533



GTC AGA CCG CCC GC









a Underlined nucleotides indicate the molecular beacon's stem.


b Sequence from databases.


c These sequences were aligned to derive the corresponding primer.


d The nucleotide positions refer to the P. aeruginosa tuf sequence fragment


(SEQ ID NO. 153)













Fungal species:
Candida lustaniae




2115b
5′-GCG GGC GGT AAG TCC ACC GGT AAG ACC
424
304-330



TTG TTG GCC CGC




 


2116 
5′-GCG GGC GTA AGT CAC CGG TAA GAC CTT
424
476-502



GTT GGC CCG C




 


2117 
5′-GCG GGC GAC GCC ATT GAG CCA CCT TCG
424
512-535



AGA GCC CGC




 


Fungal species:
Candida parapsilosis




2118b
5′-GCG GGC TCC TTG ACA ATT TCT TCG TAT
426
301-330



CTG TTC TTG GCC CGC




 


Fungal species:
Candida tropicalis




2119 
5′-GCG GGC TTA CAA CCC TAA GGC TGT TCC
429
357-384



ATT CGT TGC CCG C




 


2120 
5′-GCG GGC AGA AAC CAA GGC TGG TAA GGT
429
459-487



TAC CGG AGC CCG C




 


Fungal species:
Cryptococcus neoformans




2106 
5′-GCG AGC AGA GCA CGC CCT CCT CGC CGC
623, 1985, 1986c
 226-244d



TCG C




 


2107 
5′-GCG AGC TCC CCA TCT CTG GTT GGC ACG
623, 1985, 1986c
 390-408d



CTC GC




 


Bacterial genus:
Legianella sp.




2083 
5′-CCG CCG ATG TTC CGT AAA TTA CTT GAI
 111-112d
 488-519e



GAA GGT CGA GCC GGC GG









a Underlined nucleotides indicate the molecular beacon's stem.


b This sequence is from the complementary DNA strand of the sequence of the


originating fragment given in the Sequence Listing.


c These sequences were aligned to derive the corresponding primer.


d The nucleotide positions refer to the C. neoformans tuf (EF-1) sequence


fragment (SEQ ID NO. 623)


e The nucleotide positions refer to the L. pneumophila tuf (EF-1) sequence


fragment (SEQ ID NO. 112)













Fungal genus:
Candida sp.




2108 
5′-GCG GGC AAC TTC RTC AAG AAG GTT GGT
414, 417, 422,
 52-80c



TAC AAC CCG CCC GC
424, 426, 429, 624b



 


2109 
5′-GCG GGC CCA ATC TCT GGT TGG AAY GGT
Same as
 100-125c



GAC AAG CCC GC
SEQ ID NO. 2108



 


Bacterial group:
Pseudomonads




2121 
5′-CGA CCG CIA GCC GCA CAC CAA GTT CCG
153-155, 205,
 598-616e



GTC G
880, 2137d, 2138d,b





aUnderlined nucleotides indicate the molecular beacon's stem.


bThese sequences were aligned to derive the corresponding primer.


cThe nucleotide positions refer to the C. albicans tuf (EF-1) sequence fragment (SEQ ID NO. 624).


dSequence from databases.


eThe nucleotide positions refer to the P. aeruginosa tuf sequence fragment (SEQ ID NO. 153).













TABLE 86







Molecular beacon internal hybridization probes for


specific detection of ddl and intl gene sequences.











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequencea
SEQ ID NO.
position





Bacterial species:
E. faecium (ddl)




1237
5′-GCG AGC CGC GAA ATC GAA GTT GCT GTA
1242b
334-359



TTA GGG CTC GC




 


Bacterial species:
E. faecalis (mtl)




1238
5′-GCG AGC GGC GTT AAT TTT GGC ACC GAA
1243b
631-656



GAA GAG CTC GC





aUnderlined nucleotides indicate the molecular beacon's stem.


bSequence from databases.













TABLE 87







Internal hybridization probe for specific detection


of S. aureus sequences of unknown coding potential.











Originating DNA fragment











Nucleotide




SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Bacterial species:
Staphylococcus aureus




1234
5′-ACT AAA TAA ACG CTC ATT CG
1244
35-54
















TABLE 88







Specific and ubiquitous primers for nucleic acid amplification


(antimicrobial agents resistance genes sequences).











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Resistance gene:
aac(2′)-Ia




1344 
5′-AGC AGC AAC GAT GTT ACG CAG CAG
1348a
163-186


1345b
5′-CCC GCC GAG CAT TTC AAC TAT TG
1348a
392-414


 


1346 
5′-GAT GTT ACG CAG CAG GGC AGT C
1348a
172-193


1347b
5′-ACC AAG CAG GTT CGC AGT CAA GTA
1348a
467-490


 


Resistance gene:
aac(3′)-Ib




1349 
5′-CAG CCG ACC AAT GAG TAT CTT GCC
1351a
178-201


1350b
5′-TAA TCA GGGCAG TTG CGA CTC CTA
1351a
356-379


 


Resistance gene:
aac(3′)-IIb




1352 
5′-CCA CGC TGA CAG AGC CGC ACC G
1356a
383-404


1353b
5′-GGC CAG CTC CCA TCG GAC CCT G
1356a
585-606


 


1354 
5′-CAC GCT GAC AGA GCC GCA CCG
1356a
384-404


1355b
5′-ATG CCG TTG CTG TCG AAA TCC TCG
1356a
606-629


 


Resistance gene:
aac(3′)-IVa




1357 
5′-GCC CAT CCA TTT GCC TTT GC
1361a
295-314


1358b
5′-GCG TAC CAA CTT GCC ATC CTG AAG
1361a
517-540


 


1359 
5′-TGC CCC TGC CAC CTC ACT C
1361a
356-374


1360b
5′-CGT ACC AAC TTG CCA TCC TGA AGA
1361a
516-539


 


Resistance gene:
aac(3′)VIa




1362 
5′-CGC CGC CAT CGC CCA AAG CTG G
1366a
285-306


1363b
5′-CGG CAT AAT GGA GCG CGG TGA CTG
1366a
551-574


 


1364 
5′TTT CTC GCC CAC GCA GGA AAA ATC
1366a
502-525


1365b
5′-CAT CCT CGA CGA ATA TGC CGC G
1366a
681-702


 


Resistance gene:
aac(6′)-Ia




1367 
5′-CAA ATA TAC TAA CAG AAG CGT TCA
1371a
56-79


1368b
5′-AGG ATC TTG CCA ATA CCT TTA T
1371a
269-290


 


1379 
5′-AAA CCT TTG TTT CGG TCT GCT AAT
1371a
153-176


1380b
5′-AAG CGA TTC CAA TAA TAC CTT GCT
1371a
320-343


 


Resistance gene:
aac(6′)-Ic




1372 
5′-GCT TTC GTT GCC TTT GCC GAG GTC
1376a
157-180


1373b
5′-CAC CCC TGT TGC TTC GCC CAC TC
1376a
304-326


 


1374 
5′-AGA TAT TGG CTT CGC CGC ACC ACA
1376a
104-127


1375b
5′-CCC TGT TGC TTC GCC CAC TCC TG
1376a
301-323


 


Resistance gene:
ant(3′)-Ia




1377 
5′-GCC GTG GGT CGA TGT TTG ATG TTA
1381a
100-123


1378b
5′-GCT CGA TGA CGC CAA CTA CCT CTG
1381a
221-244


 


1379 
5′-AGC AGC AAC GAT GTT ACG CAG CAG
1381a
127-150


1380b
5′-CGC TCG ATG ACG CCA ACT ACC TCT
1381a
222-245


 


Resistance gene:
ant(4′)-Ia




1382 
5′-TAG ATA TGA TAG GCG GTA AAA AGC
1386a
149-172


1383b
5′-CCC AAA TTC GAG TAA GAG GTA TT
1386a
386-408


 


1384 
5′-GAT ATG ATA GGC GGT AAA AAG C
1386a
151-172


1385b
5′-TCC CAA ATT CGA GTA AGA GGT A
1386a
388-409


 


Resistance gene:
aph(3′)-Ia




1387 
5′-TTA TGC CTC TTC CGA CCA TCA AGC
1391a
233-256


1338b
5′-TAC GCT CGT CAT CAA AAT CAC TCG
1391a
488-511


 


1389 
5′-GAA TAA CGG TTT GGT TGA TGC GAG
1391a
468-491


1390b
5′-ATG GCA AGA TCC TGG TAT CGG TCT
1391a
669-692


 


Resistance gene:
aph(3′)-IIa




1392 
5′-TGG GTG GAG AGG CTA TTC GGC TAT
1396a
43-66


1393b
5′-CAG TCC CTT CCC GCT TCA GTG AC
1396a
250-272


 


1394 
5′-GAC GTT GTC ACT GAA GCG GGA AGG
1396a
244-267


1395b
5′-CTT GGT GGT CGA ATG GGC AGG TAG
1396a
386-409


 


Resistance gene:
aph(3′)-IIIa




1397 
5′-GTG GGA GAA AAT GAA AAC CTA T
1401a
103-124


1398b
5′-ATG GAG TGA AAG AGC CTG AT
1401a
355-374


 


1399 
5′-ACC TAT GAT GTG GAA CGG GAA AAG
1401a
160-183


1400b
5′-CGA TGG AGT GAA AGA GCC TGA TG
1401a
354-376


 


Resistance gene:
aph(3′)VIa




1402 
5′-TAT TCA ACA ATT TAT CGG AAA CAG
1406a
18-41


1403b
5′-TCA GAG AGC CAA CTC AAC ATT TT
1406a
175-197


 


1404 
5′-AAA CAG CGT TTT AGA GCC AAA TAA
1406a
36-59


1405b
5′-TTC TCA GAG AGC CAA CTC AAC ATT
1406a
177-200


 


Resistance gene:
blaCARB




1407 
5′-CCC TGT AAT.AGA AAA GCA AGT AGG
1411a
351-374


1408b
5′-TTG TCG TAT CCC TCA AAT CAC C
1411a
556-577


 


1409 
5′-TGG GAT TAC AAT GGC AAT CAG CG
1411a
205-227


1410b
5′-GGG GAA TAG GTC ACA AGA TCT GCT T
1411a
329-353


 


Resistance gene:
blaCMY2




1412 
5′-GAG AAA ACG CTC CAG CAG GGC
1416a
793-813


1413b
5′-CAT GAG GCT TTC ACT GCG GGG
1416a
975-995


 


1414 
5′-TAT CGT TAA TCG CAC CAT CAC
1416a
90-110


1415b
5′-ATG CAG TAA TGC GGC TTT ATC
1416a
439-459


 


Resistance genes:
blaCTX-M-1, blaCTX-M-2




1417 
5′-TGG TTA ACT AYA ATC CSA TTG CGG A
1423a
314-338


1418b
5′-ATG CTT TAC CCA GCG TCA GAT T
1423a
583-604


 


Resistance gene:
blaCTX-M-1




1419 
5′-CGA TGA ATA AGC TGA TTT CTC ACG
1423a
410-433


1420b
5′-TGC TTT ACC CAG CGT CAG ATT ACG
1423a
580-603


 


1421 
5′-AAT TAG AGC GGC AGT CGG GAG GAA
1423a
116-139


1422b
5′-GAA ATC AGC TTA TTC ATC GCC ACG
1423a
405-428


 


Resistance gene:
blaCTX-M-2




1424 
5′-GTT AAC GGT GAT GGC GAC GCT AC
1428a
30-52


1425b
5′-GAA TTA TCG GCG GTG TTA ATC AGC
1428a
153-176


 


1426 
5′-CAC GCT CAA TAC CGC CAT TCC A
1428a
510-531


1427b
5′-TTA TCG CCC ACT ACC CAT GAT TTC
1428a
687-710


 


Resistance gene:
blaIMP




1429 
5′-TTT ACG GCT AAA GAT ACT GAA AAG T
1433a
205-229


1430b
5′-GTT TAA TAA AAC AAC CAC CGA ATA AT
1433a
513-538


 


1431 
5′-TAA TTG ACA CTC CAT TTA CGG CTA A
1433a
191-215


1432b
5′-ACC GAA TAA TAT TTT CCT TTC AGG CA
1433a
497-522


 


Resistance gene:
blaOXA2




1434 
5′-CAC AAT CAA GAC CAA GAT TTG CGA T
1438a
319-343


1435b
5′-GAA AGG GCA GCT CGT TAC GAT AGA G
1438a
532-556


 


Resistance gene:
blaOXA10




1436 
5′-CAG CAT CAA CAT TTA AGA TCC CCA
1439a
194-217


1437b
5′-CTC CAC TTG ATT AAC TGC GGA AAT TC
1439a
479-504


 


Resistance gene:
blaPER-1




1440 
5′-AGA CCG TTA TCG TAA ACA GGG CTA AG
1442a
281-306


1441b
5′-TTT TTT GCT CAA ACT TTT TCA GOA TC
1442a
579-604


 


Resistance gene:
blaPE.R2




1443 
5′-CTT CTG CTC TGC TGA TGC TTG GC
1445a
32-54


1444b
5′-GGC GAC CAG GTA TTT TGT AAT ACT GC
1445a
304-329


 


Resistance genes:
blaPER1, blaPER2




1446 
5′-GGC CTG YGA TTT GTT ATT TGA ACT GGT
1442a
414-440


1447b
5′-CGC TST GGT CCT GTG GTG GTT TC
1442a
652-674


 


1448 
5′-GAT CAG GTG CAR TAT CAA AAC TGG AC
1442a
532-557


1449b
5′-AGC WGG TAA CAA YCC TTT TAA CCG CT
1442a
671-696


 


Resistance gene:
blaSHV




1883 
5′-AGC CGC TTG AGC AAA TTA AAC TA
1900a
71-93


1884b
5′-GTA TCC CGC AGA TAA ATC ACC AC
1900a
763-785


 


1885 
5′-AGC GAA AAA CAC CTT GCC GAC
1900a
313-333


1884b
5′-GTA TCC CGC AGA TAA ATC ACC AC
1900a
763-785


 


Resistance gene:
blaTEM




1906 
5′-CCT TAT TCC CTT TTT TGC GG
1927a
27-46


1907b
5′-CAC CTA TCT CAG CGA TCT GTC T
1927a
817-838


 


1908 
5′-AAC AGC GGT AAG ATC CTT GAG AG
1927a
148-170


1907b
5′-CAC CTA TCT CAG CGA TCT GTC T
1927a
817-838


 


Resistance gene:
catI




2145 
5′-GCA AGA TGT GGC GTG TTA CGG T
2147a
363-384


2146b
5′-GGG GCG AAG AAG TTG TCC ATA TT
2147a
484-506


 


Resistance gene:
catII




2148 
5′-CAG ATT AAA TGC GGA TTC AGC C
2150a
67-88


2149b
5′-ATC AGG TAA ATC ATC AGC GGA TA
2150a
151-173


 


Resistance gene:
catIII




2151 
5′-ATA TTT CAG CAT TAC CTT GGG TT
2153a
419-441


2152b
5′-TAC ACA ACT CTT GTA GCC GAT TA
2153a
603-625


 


Resistance gene:
catP




2154 
5′-CGC CAT TCA GAG TTT AGG AC
2156a
178-197


2155b
5′-TTC CAT ACC GTT GCG TAT CAC TT
2156a
339-361


 


Resistance gene:
cat




2157 
5′-CCA CAG AAA TTG ATA TTA GTG TTT TAT
2159a
89-115


2158b
5′-TCG CTA TTG TAA CCA GTT CTA
2159a
201-221


 


2160 
5′-TTT TGA ACA CTA TTT TAA CCA GC
2162a
48-70


2161b
5′-GAT TTA ACT TAT CCC AAT AAC CT
2162a
231-253


 


Resistance gene:
dfrA




1450 
5′-ACC ACT GGG AAT ACA CTT GTA ATG GC
1452a
106-131


1451b
5′-ATC TAC CTG GTC AAT CAT TGC TTC GT
1452a
296-321


 


Resistance gene:
dhfrIa




1457 
5′-CAA AGG TGA ACA GCT CCT GTT T
1461a
75-96


1458b
5′-TCC GTT ATT TTC TTT AGG TTG GTT AAA
1461a
249-275


 


1459 
5′-AAG GTG AAC AGC TCC TGT TT
1461a
77-96


1560b
5′-GAT CAC TAC GTT CTC ATT GTC A
1461a
207-228


 


Resistance genes:
dhfrIa, dhfrXV




1453 
5′-ATC GAA GAA TGG AGT TAT CGG RAA TG
1461a
27-52


1454b
5′-CCT AAA AYT RCT GGG GAT TTC WGG A
1461a
384-408


 


1455 
5′-CAG GTG GTG GGG AGA TAT ACA AAA
1461a
290-313


1456b
5t-TAT GTT AGA SRC GAA GTC TTG GKT AA
1461a
416-441


 


Resistance gene:
dbfrIb




1466 
5′-AAG CAT TGA CCT ACA ATC AGT GT
1470a
 98-120


1467b
5′AAT ACA ACT ACA TTG TCA TCA TTT GAT
1470a
204-230


 


1468 
5′-CGT TAC CCG CTC AGG TTG GAC ATC AA
1470a
183-208


1469b
5′-CAT CCC CCT CTG GCT CGA TGT CG
1470a
354-376


 


Resistance gene:
dbfrV




1471 
5′-GAT AAT GAC AAC GTA ATA GTA TTC CC
1475a
208-233


1472b
5′-GCT CAA TAT CAA TCG TCG ATA TA
1475a
342-364


 


1473 
5′-TTA AAG CCT TGA CGT ACA ACC AGT GG
1475a
 95-120


1474b
5′-TGG GCA ATG TTT CTC TGT AAA TCT CC
1475a
300-325


 


Resistance genes:
dhfrIb, dhfrV




1462 
5′-GCA CTC CCY AAT AGG AAA TAC GC
1470a
157-179


1463b
5′-AGT GTT GCT CAA AAA CAA CTT CG
1470a
405-427


 


1464 
5′-ACG TTY GAA TCT ATG GGM GCA CT
1470a
139-161


1465b
5′-GTC GAT AAG TGG AGC GTA GAG GC
1470a
328-350


 


Resistance gene:
dhfrVI




1476 
5′-GGC GAG CAG CTC CTA TTC AAA G
1480a
 79-100


1477b
5′-TAG GTA AGC TAA TGC CGA TTC AAC A
1480a
237-261


 


1478 
5′-GAG AAT GGA GTA ATT GGC TCT GGA TT
1480a
31-56


1479b
5′-GCG AAA TAC ACAACA TCA GGG TCA T
1480a
209-233


 


Resistance gene:
dhfrVII




1485 
5′-AAA ATG GCG TAA TCG GTA ATG GC
1489a
32-54


1486b
5′-CAT TTG AGC TTG AAA TTC CTT TCC TC
1489a
189-214


 


1487 
5′-AAT CGA AAA TAT GCA GTA GTG TCG AG
1489a
166-191


1488b
5′-AGA CTA TTG TAG ATT TGA CCG CCA
1489a
294-317


 


Resistance genes:
dhfrVII, dhfrXVII




1481 
5′-RTT ACA GAT CAT KTA TAT GTC TCT
1489a
268-291


1482b
5′-TAA TTT ATA TTA GAC AWA AAA AAC TG
1489a
421-446


 


1483 
5-CAR YGT CAG AAA ATG GCG TAA TC
1489a
23-45


1484b
5′-TKC AAA GCR WTT TCT ATT GAA GGA AA
1489a
229-254


 


Resistance gene:
dhfrVIII




1490 
5′-GAC CTA TGA GAG CTT GCC CGT CAA A
1494a
144-168


1491b
5′-TCG CCT TCG TAC AGT CGC TTA ACA AA
1494a
376-401


 


1492 
5′-CAT TTT AGC TGC CAC CGC CAA TGG TT
1494a
18-43


1493b
5′-GCG TCG CTG ACG TTG TTC ACG AAG A
1494a
245-269


 


Resistance gene:
dbfrIX




1495 
5′-TCT CTA AAC ATG ATT GTC GCT GTC
1499a
 7-30


1496b
5′-CAG TGA GGC AAA AGT TTT TCT ACC
1499a
133-156


 


1497 
5′-CGG ACG ACT TCA TGT GGT AGT CAG T
1499a
171-195


1498b
5′-TTT GTT TTC AGT AAT GGT CGG GAC CT
1499a
446-471


 


Resistance gene:
dhfrXII




1500 
5′-ATG GGG TTA TTG GCA ATG GTG CTA
1504a
50-73


1501b
5′-GCG GTA GTT AGC TTG GGG TGA GAT T
1504a
201-225


 


1502 
5′-GGG GGC GGA GCT GAG ATA TAC A
1504a
304-325


1503b
5′-AAC GGA GTG GGT GTA CGG AAT TAG AG
1504a
452-477


 


Resistance gene:
dhfzXIII




1505 
5′-ATT TTT CGC AGG GTG ACG GAG AGG
1507a
106-129


1506b
5′-GGG ATG AGA GAA CGT GGA ATT CTG GTG
1507a
413-439


 


Resistance gene:
dbfrXV




1508 
5′-AGA ATG TAT TGG TAT TTG GAT GTA TGG
1512a
215-241


1509b
5′-GAA TGT GGA TTG TTG AAA TAT GTA AA
1512a
336-361


 


1510 
5′-TGG AGT GGC AAA GGG GAA GAA T
1512a
67-88


1511b
5′-GAG ACA GAA TCA GAT GAT CGG TTA TGG
1512a
266-292


 


Resistance gene:
dhfrXVII




1513 
5′-TTG AAG GTG AAA TGA AAA CGT CC
1517a
201-223


1514b
5′-GAA ATT GTG AGG GAT TAT AGG GAA T
1517a
381-405


 


1515 
5′-GTG GTG AGT AAA AGG TGA GCA AC
1517a
66-88


1516b
5′-TGT TTG AAA GGA TTT TGT ATT GAA GG
1517a
232-257


 


Resistance gene:
embB




2102 
5′-GAC GTT CAC CGT GAG GGA GG
2105a
822-841


2103b
5′-CGA ACG AGG GGA AAT AGT TGG AC
2105a
948-970


 


Resistance genes:
ereA, ereA2




1528 
5′-AAC TTG AGG GAT TTT GGG ATA CCC TG
1530a
 80-105


1529b
5′-TTG CGG ATG AAA TAA CGG CGG ACT
1530a
317-340


 


Resistance gene:
ereB




1531 
5′-TCT TTT TGT TAC GAC ATA CGC TTT T
1535a
152-176


1532b
5′-AGT GCT TCT TTA TCC GCT GTT CTA
1535a
456-479


 


1533 
5′-CAG CGG ATA AAG AAG CAC TAC ACA TT
1535a
461-486


1534b
5′-CCT CCT GAA ATA AAG CCC GAC AT
1535a
727-749


 


Resistance gene:
gyrA




1340 
5′-GAA CAA GGT ATG ACA CCG GAT AAA T
1299a
163-188


1341b
5′-GAT AAC TGA AAT CCT GAG CCA TAC G
1299a
274-299


 


1936 
5′-TAC CAC CCG CAC GGC
1954a
205-219


1937b
5′-CGG AGT CGC CGT CGA TG
1954a
309-325


 


1942 
5′-GAC TGG AAC AAA GCC TAT AAA AAA TCA
1954a
148-174


1937b
5′-CGG AGT CGC CGT CGA TG
1954a
309-325


 


2040 
5′-TGT GAC CCC AGA CAA ACC C
2054a
33-51


2041b
5′-GTT GAG CGG CAG CAC TAT CT
2054a
207-226


 


Resistance gene:
inhA




2098 
5′-CTG AGT CAC ACC GAC AAA CGT C
2101a
910-931


2099b
5′-CCA GGA CTG AAC GGG ATA CGA A
2101a
1074-1095


 


Resistance genes:
linA, linA′




1536 
5′-AGA TGT ATT AAC TGG AAA ACA ACA A
1540a
 99-123


1537b
5′-CTT TGT AAT TAG TTT CTG AAA ACC A
1540a
352-376


 


1538 
5′-TTA GAA GAT ATA GGA TAC AAA ATA GAA G
1540a
187-214


1539b
5′-GAA TGA AAA AGA AGT TGA GCT T
1540a
404-425


 


Resistance gene:
linB




1541 
5′-TGA TAA TCT TAT ACG TGG GGA ATT T
1545a
246-270


1542b
5′-ATA ATT TTC TAA TTG CCC TGT TTC AT
1545a
359-384


 


1543 
5′-GGG CAA TTA GAA AAT TAT TTA TCA GA
1545a
367-392


1544b
5′-TTT TAC TCA TGT TTA GCC AAT TAT CA
1545a
579-604


 


Resistance gene:
mefA




1546 
5′-CAA GAA GGA ATG GCT GTA CTA C
1548a
625-646


1547b
5′-TAA TTC CCA AAT AAC CCT AAT AAT AGA
1548a
816-842


 


Resistance gene:
mefE




1549 
5′-GCT TAT TAT TAG GAA GAT TAG GGG GC
1551a
815-840


1550b
5′-TAG CAA GTG ACA TGA TAC TTC CGA
1551a
1052-1075


 


Resistance genes:
mefA, mefE




1552 
5′-GGC AAG CAG TAT CAT TAA TCA CTA
1548a
50-73


1553b
5′-CAA TGC TAC GGA TAA ACA ATA CTA TC
1548a
318-343


 


1554 
5′-AGA AAA TTA AGC CTG AAT ATT TAG GAC
1548a
1010-1035


1555b
5′-TAG TAA AAA CCA ATG ATT TAC ACC G
1548a
1119-1143


 


Resistance genes:
mphA, mphK




1556 
5′-ACT GTA CGC ACT TGC AGC CCG ACA T
1560a
33-57


1557b
5′-GAA CGG CAG GCG ATT CTT GAG CAT
1560a
214-237


 


1558 
5′-GTG GTG GTG CAT GGC GAT CTC T
1560a
583-604


1559b
5′-GCC GCA GCG AGG TAC TCT TCG TTA
1560a
855-878


 


Resistance gene:
mupA




2142 
5′-GCC TTA ATT TCG GAT AGT GC
2144a
1831-1850


2143b
5′-GAG AAA GAG CCC AAT TAT CTA ATG T
2144a
2002-2026


 


Resistance gene:
parC




1342 
5′-GAT GTT ATT GGT CAA TAT CAT CCA
1321a
205-229


1343b
5′AAG AAA CTG TCT CTT TAT TAA TAT CAC GT
1321a
396-425


 


1934 
5′-GAA CGC CAG CGC GAA ATT CAA AAA G
1781
67-91


1935b
5′-AGC TCG GCA TAC TTC GAC AGG
1781
277-297


 


2044 
5′-ACC GTA AGT CGG CCA AGT CA
2055a
176-195


2045b
5′-GTT CTT TCT CCG TAT CGT C
2055a
436-454


 


Resistance gene:
ppflo-like




2163 
5′-ACC TTC ATC CTA CCG ATG TGG GTT
2165a
922-945


2164b
5′-CAA CGA CAC CAG CAC TGC CAT TG
2165a
1136-1158


 


Resistance gene:
rpoB




2065 
5′-CCA GGA CGT GGA GGC GAT CAC A
2072a
1218-1239


2066b
5′-CAC CGA CAG CGA GCC GAT CAG A
2072a
1485-1506


 


Resistance gene:
satG




1581 
5′-AAT TGG GGA CTA CAC CTA TTA TGA TG
1585a
 93-118


1582b
5′-GGC AAA TCA GTC AGT TCA GGA GT
1585a
310-332


 


1583 
5′-CGA TTG GCA ACAATA CAC TCC TG
1585a
294-316


1584b
5′-TCA CCT ATT TTT ACG CCT GGT AGG AC
1585a
388-413


 


Resistance gene:
sulII




1961 
5′-GCT CAA GGC AGA TGG CAT TCC C
1965a
222-243


1962b
5′-GGA CAA GGC GGT TGC GTT TGA T
1965a
496-517


 


1963 
5′-CAT TCC CGT CTC GCT CGA CAG T
1965a
237-258


1964b
5-ATC TGC CTG CCC GTC TTG C
1965a
393-411


 


Resistance gene:
tetB




1966 
5′-CAT GCC AGT CTT GCC AAC G
1970a
66-84


1967b
5′-CAG CAA TAA GTA ATC CAG CGA TG
1970a
242-264


 


1968 
5′-GGA GAG ATT TCA CCG CAT AG
1970a
457-476


1969b
5′-AGC CAA CCA TCA TGC TAT TCC A
1970a
721-742


 


Resistance gene:
tetM




1586 
5′-CCC ACA ATC TTT TTT ATC AAT AA
1590a
361-386


1587b
5′-CAT TGT TCA GAT TCG GTA AAG TTC
1590a
501-524


 


1588 
5′-GTT TTT GAA GTT AAA TAG TGT TCT T
1590a
957-981


1589b
5′-CTTCCA TTT GTA CTT TCC CTA
1590a
1172-1192


 


Resistance gene:
vatB




1609 
5′-GCC CTG ATC CAA ATA GCA TAT A
1613a
11-32


1610b
5′-CCT GGC ATA ACA GTA ACA TTC TG
1613a
379-401


 


1611 
5′-TGG GAA AAA GCA ACT CCA TCT C
1613a
301-322


1612b
5′-ACA ACT GAA TTC GCA GCA ACA AT
1613a
424-446


 


Resistance gene:
vatC




1614 
5′-CCA ATC CAG AAG AAA TAT ACC C
1618a
26-47


1615b
5′-ATT AGT TTA TCC CCA ATC AAT TCA
1618a
177-200


 


1616 
5′-ATA ATG AAT GGG GCT AAT CAT CGT AT
1618a
241-266


1617b
5′-GCC AAC AAC TGA ATA AGG ATC AAC
1618a
463-486


 


Resistance gene:
vga




1619 
5′-AAG GCA AAA TAA AAG GAG CAA AGC
1623a
641-664


1620b
5′-TGT ACC CGA GAC ATC TTC ACC AC
1623a
821-843


 


1621 
5′-AAT TGA AGG ACG GGT ATT GTG GAA AG
1623a
843-868


1622b
5′-CGA TTT TGA CAG ATG GCG ATA ATG AA
1623a
975-1000


 


Resistance gene:
vgaB




1624 
5′-TTC TTT AAT GCT COT AGA TGA ACC TA
1628a



1625b
5′-TTT TCG TAT TCT TCT TGT TGC TTT C
1628a
578-602


 


1626 
5′-AGG AAT GAT TAA GCC CCC TTC AAA AA
1628a
663-688


1627b
5′-TTA CAT TGC GAC CAT GAA ATT GCT CT
1628a
849-874


 


Resistance genes:
vgb, vgh




1629 
5′-AAG GGG AAA GTT TGG ATT ACA CAA CA
1633a
73-98


1630b
5-GAA CCA CAG GGC ATT ATC AGA ACC
1633a
445-468


 


1631 
5′-CGA CGA TGC TTT ATG GTT TGT
1633a
576-596


1632b
5′-GTT AAT TTG CCT ATC TTG TCA CAC TC
1633a
850-875


 


Resistance gene:
vgbB




1634 
5′-TTA ACT TGT CTA TTC CCG ATT CAG G
1882a
23-47


1635b
5′-GCT GTG GCA ATG GAT ATT CTG TA 1882a
267-289



 


1636 
5′-TTC CTA CCC CTG ATG CTA AAG TGA
1882a
155-178


1637b
5′-CAA AGT GCG TTA TCC GAA CCT AA
1882a
442-464


 







Sequencing primers










Resistance gene:
gyrA




1290 
5′-GAY TAY GCI ATG 151 GTI ATH GT 1299a
70-83



1292b
5′-ARI SCY TCI ARI ATR TGI GC
1299a
1132-1152


 


1291 
5′-GCI YTI CCI GAY GTI MGI GAY GG
1299a
100-123


1292b
5′-ARI SCY TCI ARI ATR TGI GC
1299a
1132-1152


 


1293 
5′-ATG GCT GAA TTA CCT CAA TC
1299a
 1-21


1294b
5′-ATG ATT GTT GTA TAT CTT CTT CAA C
1299a
2626-2651


 


1295b
5′-CAG AAA GTT TGA AGC GTT GT
1299a
1255-1275


 


1296 
5′-AAC GAT TCG TGA GTC AGA TA
1299a
1188-1208


 


1297 
5′-CGG TCA ACA TTG AGG AAG AGC T
1300a
29-51


1298b
5′-ACG AAA TCG ACC GTC TCT TTT TC
1300a
415-437


 


Resistance gene:
gyrB




1301 
5′-GTI MGI AWl MGI CCI GSI ATG TA
1307a
 82-105


1302b
5′-TAI ADI GGI GGI KKI GCI ATR TA
1307a
1600-1623


 


1303 
5′-GGI GAI GAI DYI MGI GAR GG
1307a
944-975


1304b
5′-CIA RYT TIK YIT TIG TYT G
1307a
1024-1043


 


1305 
5′-ATG GTG ACT GCA TTG TCA GAT G
1307a
 1-23


1306b
5′-GTC TAC GGT TTT CTA CAA CGT C
1307a
1858-1888


 


Resistance gene:
parC




1308 
5′-ATG TAY GTI ATI ATG GAY MGI GC
67-90



1309b
5′-ATI ATY TTR TTI CCY TTI CCY TT
1993-2016



 


1310 
5′-ATI ATI TSI ATI ACY TCR TC
1320a
1112-1132


1311b
5′-GAR ATG AAR ATI MGI GGI GAR CA
1320a
1288-1311


 


1312 
5′-AAR TAY ATI ATI CAR GAR MGI GC
1321a
67-90


1313b
5′-AMI AYI CKR TGI GGI TTI TTY TT
1321a
2212-2235


 


1314 
5′-TAI GAl TTY ACI GAl SMI CAR GC
1321a
1228-1251


1315b
5′-ACI ATI GCI TCI GCY TGI KSY TC
1321a
1240-1263


 


1316 
5′-GTG AGT GAA ATA ATT CAA GAT T
1321a
 1-23


1317b
5′-CAC CAA AAT CAT CTG TAT CTA C
1321a
2356-2378


 


1318 
5′-ACC TAY TCS ATG TAC GTR ATC ATG GA
1320a
58-84


1319b
5′-AGR TCG TCI ACC ATC GGY AGY TT
1320a
832-855


 


Resistance gene:
parE




1322 
5′-RTI GAl AAY ISI GTI GAY GAR G
1328a
133-155


1325b
5′-RTT CAT YTC ICC IAR ICC YTT
1328a
1732-1752


 


1323 
5′-ACI AWR SAl GGI GGI ACI CAY G
1328a
829-850


1324b
5′-CCI CCI GCI SWR TCI CCY TC
1328a
1280-1302


 


1326 
5′-TGA TTC AAT ACA GGT TTT AGA G
1328a
27-49


1327b
5′-CTA GAT TTC CTC CTC ATC AAA T
1328a
1971-1993





aSequence from databases.


bThese sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.













TABLE 89







Internal hybridization probes for specific detection


of antimicrobial agents resistance genes sequences.











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position













Resistance gene:
aph3′VIa




2252
5′-CCA CAT ACA GTG TCT CTC
1406a
149-166


 


Resistance gene:
blaSBV




1886
5′-GAC GCC CGC GCC ACC ACT
1900a
484-501


 


1887
5′-GAC GCC CGC GAC ACC ACT A
1899a
514-532


 


1888
5′-GAC GCC CGC AAC ACC ACT A
1901a
514-532


 


1889
5′-GTT CGC AAC TGC AGC TGC TG
1899a
593-612


 


1890
5′-TTC GCA ACG GCA GCT GCT G
1899a
594-612


 


1891
5′-CCG GAG CTG CCG AIC GGG
1902a
692-709


 


1892
5′-CGG AGC TGC CAA RCG GGG
1903a
693-710


 


1893
5′-GGA GCT GGC GAR CGG GGT
1899a
694-711


 


1894
5′-GAC CGG AGC TAG CGA RCG
1904a
690-707


 


1895
5′-CGG AGC TAG CAA RCG GGG T
1899a
693-711


 


1896
5′-GAA ACG GAA CTG AAT GAG GCG
1899a
484-504


 


1897
5′-CAT TAC CAT GGG CGA TAA CAG
1899a
366-386


 


1898
5′-CCA TTA CCA TGA GCG ATA ACAG
1899a
365-386


 


Resistance gene:
blaTEM




1909
5′-ATG ACT TGG TTA AGT ACT CAC C
1928a
293-314


 


1910
5′-ATG ACT TGG TTG AGT ACT CAC C
1927a
293-314


 


1911
5′-CCA TAA CCA TGG GTG ATA ACA C
1928a
371-392


 


1912
5′-CCA TAA CCA TGA GTG ATA ACA C
1927a
371-392


 


1913
5′-CGC CTT GAT CAT TGG GAA CC
1928a
475-494


 


1914
5′-CGC CTT GAT CGT TGG GAA CC
1927a
475-494


 


1915
5′-CGC CTT CAT AGT TGG GAA CC
1929a
475-494


 


1916
5′-CGT GGG TCT TGC GGT ATC AT
1927a
712-731


 


1917
5′-CGT GGG TCT GGC GGT ATC AT
1930a
712-731


 


1918
5′-GTG GGT CTC ACG GTA TCA TTG
1927a
713-733


 


1919
5′-CGT GGG TCT CTC GGT ATC ATT
1931a
712-732


 


1920
5′-CGT GGI TCT CGC GGT ATC AT
1927a
712-731


 


1921
5′-CGT GGG TCT AGC GGT ATC ATT
1932a
713-733


 


1922
5′-GTT TTC CAA TGA TTA GCA CTT TTA
1927a
188-211


 


1923
5′-GTT TTC CAA TGA TAA GCA CTT TTA
1927a
188-211


 


1924
5′-GTT TTC CAA TGC TGA GCA CTT TT
1932a
188-210


 


1925
5′-CGT TTT CCA ATG ATG AGC ACT TT
1927a
187-209


 


1926
5′-GTT TTC CAA TGG TGA GCA CTT TT
1933a
188-210


 


2006
5′-TGG AGC CGG TGA GCG TGG
1927a
699-716


 


2007
5′-TGG AGG GAG TGA GCG TGG
2010a
699-716


 


2008
5′-TGT GGA GCG GAT GAG CGT G
1929a
697-715


 


2009
5′-GTG GAG CGA GTA AGC GTG G
2011a
698-716


 


2141
5′-GAG GAG TGA GAG AAA AGC
1927a
311-328


 


Resistance gene:
dbfrIa




2253
5′-GAT TAG CGA ACC GAA AGT A
1461a
158-176


 


Resistance gene:
embB




2104
5′-GTG GGG ATG GGI GGA GTG
2105a
910-927


 


Resistance gene:
gyrA




1333
5′-TGA TGG TGA GTT ATG TAT TTA TG
1299a
240-263


 


1334
5′-GAT GTA TTT ATA AAG GAA TGG TA
1299a
251-274


 


1335
5′-GTA TTT ATG GAG GAA TGG T
1299a
254-273


 


1940
5′-GTA TGG TTG GTG AGG TAA T
1299a
206-224


 


1943
5′-GGT GGT GGA GGG GGA G
1954a
279-294


 


1945
5′-GGG GGA GTA GGG GGT AT
1954a
216-232


 


1946
S′-GGG GGA GTT GGG GGT AT
1954a
216-232


 


1947
5′-GGG TAT AGG GGA GGA TGG T
1954a
227-245


 


1948
5′-GGG GTA TAG AAG AGG ATG G
1954a
226-244


 


1949
5′-GGG TAT AGG GGA GGA TGG T
1954a
227-245


 


2042
5′-GAG GGG GAT TTG TGT ATT TA
2054a
103-122


 


2043
5′-GAG GGG GAT TAG TGT ATT TA
2054a
103-122


 


Resistance gene:
inhA




2100
5′-GGG AGA GGA TAG GTT GTG
2101a
1017-1034


 


Resistance gene:
parC




1336
5′-TGG AGA GTA GTG AGT GT
1321a
232-249


 


1337
5-TGG AGA GTT GTG AGT GT
1321a
232-249


 


1338
5′-GTG TAG GGA GGA ATG
1321a
245-260


 


1339
5′-GGA GGG GAA ATG GGT
1321a
342-357


 


1941
5′-GGA ATG GTG GGT TTA AGT
1321a
253-270


 


1944
5′-TTT GGG GGG GAT GGG TTA G
1781
247-265


 


1950
5′-GGG GAG ATG GGG TGG
1781
137-151


 


1951
5′-GGG GAG AGA GGG TGG TA
1781
137-153


 


1952
5′-CCT GCT ATG GAG CGA TGG T
1781
147-165


 


1953
5′-CGC CTG CTA TAA AGC GAT GGT
1781
145-165


 


2046
5′-ACG GGG ATT TTT CTA TCT AT
2055a
227-246


 


Resistance gene:
rpoB




2067
5-AGC TGA GCC AAT TCA TGG
2072a
1304-1321


 


2068
5′-ATT CAT GGA CCA GAA CAA C
2072a
1314-1332


 


2069
5′-CGC TGT CGG GGT TGA CCC
2072a
1334-1351


 


2070
5′-GTT GAC CCA CAA GCG CCG
2072a
1344-1361


 


2071
5-CGA CTG TCG GCG CTG GGG
2072a
1360-1377


 


Resistance gene:
tetM




2254
5′-ACC TGA ACA GAG AGA AAT G
1590a
1062-1080





aSequence from databases.













TABLE 90







Molecular beacon internal hybridization probes


for specific detection of atpD sequences.











Originating DNA fragment












SEQ ID
Nucleotide


SEQ ID NO.
Nucleotide sequencea
NO.
position













Bacterial species:
Bacteroides fragilis




2136
5′-CCA ACG CGT CCT CAA TCA TTT CTA ACT TCT
929
353-382



ATG GCC GGC GTT GG




 


Bacterial species:
Bordetella pertussis




2182
5′-GCG CGC CAA CGA CTT CTA CCA CGA AAT GGA
1672
576-605



AGA GTC GCG CGC




 


Bacterial group:
Campylobacter jejuni and C. coil




2133
5′-CCA CGC ACA WAA ACT TGT TTT AGA AGT
1576, 1600, 1849,
 44-73d



AGC AGC WCA GCG TGG
1863, 2139b, c



 


Fungal species:
Candida glabrata




2078
5′-CCG AGC CTT GGT CTT CGG CCA AAT GAA CGC
463
442-463



TCG G




 


Fungal species:
Candida krusei




2075
5′-CCG AGC CAG GTT CTG AAG TCT CTG CAT TAT
468
720-748



TAG GTG CTC GG




 


Fungal species:
Candida lusitaniae




2080
5′-CCG AGC CGA AGA GGG CCA AGA TGT CGC TCG
470
520-538



G




 


Fungal species:
Candida parapailosis




2079
5′-CCG AGC GTT CAG TTA CTT CAG TCC AAG CCG
472
837-860



GCT CGG




 


Fungal species:
Candida tropicalis




2077
5′-CCG AGC AAC CGA TCC AGC TCC AGC TAC GCT
475
877-897



CGG




 


Bacterial species:
Kiebalella pneumoniae




2281
5′-CCC CCA GCT GGG CGG CGG TAT CGA TGG GGG
317
40-59







a Underlined nucleotides indicate the molecular beacon′s stem.


b Sequence from databases:


c These sequences were aligned to derive the corresponding primer.


d The nucleotide positions refer to the C. jejuni atpD sequence fragment


(SEQ ID NO. 1576)













Fungal genus:
Candida sp.




2076
5′-CCG AGC YGA YAA CAT TTT CAG ATT CAC CCA
460-478, 663b
 697-723c



RGC GCT CGG





a Underlined nucleotides indicate the molecular beacon's stem.


b These sequences were aligned to derive the corresponding primer.


c The nucleotide positions refer to the C. albicans atpD sequence fragment (SEQ ID NO. 460)













TABLE 91







Internal hybridization probes for


specific detection of atpD sequences.











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Bacterial species:
Acinetobacter baumannii




2169
5′-CCC GTT TGC GAA AGG TGG
243
304-321


 


Bacterial species:
Klebsiella pneumoniae




2167
5′-CAG CAG CTG GGC GGC GGT
317
36-53
















TABLE 92







Internal hybridization probes for specific


detection of ddl and intl sequences.











Originating DNA fragment













Nucleotide


SEQ ID NO.
Nucleotide sequence
SEQ ID NO.
position





Bacterial species:
Enterococcus faecium (ddl)




2286
5′-AGT TGC TGT ATT AGG AAA TG
2288a
784-803


 


2287
5′-TCG AAG TTG CTG TAT TAG GA
2288a
780-799


 


Bacterial species:
Enterococcus faecalis mtl)




2289
5′-CAC CGA AGA AGA TGA AAA AA
1243a
264-283


 


2290
5′-TGG CAC CGA AGA AGA TGA
1243a
261-278


 


2291
5′-ATT TTG GCA CCG AAG AAG A
1243a
257-275





aSequence from databases.

## Claims

1. A composition capable of detecting and identifying E. coli and Shigella bacteria in a sample using a nucleic acid amplification assay, said composition comprising:
an amplification primer pair comprising a first and a second oligonucleotide, wherein said primer pair is adapted to amplify a region of the E. coli and Shigella DNA between and including said first and second oligonucleotides to produce a detectable amplification product that consists of sequences from the tuf gene, wherein said first oligonucleotide is, or is fully complementary to, SEQ ID NO: 1661 and optionally includes one degenerate nucleotide, or said first oligonucleotide consists of at least 15 contiguous nucleotides of SEQ ID NO: 1661 or the complement thereof, and wherein said second oligonucleotide is, or is fully complementary to SEQ ID NO: 1665 and optionally includes one degenerate nucleotide, or said second oligonucleotide consists of at least 15 contiguous nucleotides of SEQ ID NO: 1665 or the complement thereof.

2. The composition of claim 1, wherein said first and second oligonucleotides are SEQ ID NOs: 1661 and 1665.

3. The composition of claim 2, further comprising a probe, wherein said probe hybridizes to the amplification product under a set of conditions that is the same as the condition used in the nucleic acid amplification assay.

4. The composition of claim 3, wherein said probe comprises a detectable label.

5. The composition of claim 4, wherein said detectable label is fluorescent.

6. The composition of claim 1, further comprising a DNA polymerase.

7. A method of detecting and identifying E. coli and Shigella bacteria in a sample, comprising:
providing said sample;
contacting said sample with the composition of claim 1 under conditions that allow amplification of the region of E. coli and Shigella DNA between and including the first and second oligonucleotides in the presence of E. coli and Shigella DNA in the sample, to generate an amplification product; and
testing for the presence of the amplification product, wherein the presence of the amplification product is indicative of the presence of E. coli or Shigella in the sample.

8. The method of claim 7, wherein said testing comprises:
contacting said sample with a probe comprising a detectable label, wherein contacting the probe to the amplification product is performed under a set of conditions that is the same as the condition used in the nucleic acid amplification step; and measuring the amount of probe hybridized to the amplification product.

9. The method of claim 8, further comprising:
recording the amount of probe hybridized to the amplification product.

10. The method of claim 7, wherein said first and second oligonucleotides are SEQ ID NOs: 1661 and 1665.

11. The method of claim 8, wherein said detectable label is fluorescent.