Abstract:
A method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention relates to the field of PCR methods, and specific primers therefore, as well as their use in the identification of any type of bacteria, and in particular RNA forms of bacteria.  
       BACKGROUND OF THE INVENTION  
       [0002]     The use of biological fluids for therapeutic human application such as plasmas, albumin, live vaccines, stem cells requires that they are absolutely devoid of bacterial contamination. It has been found that filtering, and possibly other traditional methods, may fail to eliminate all forms of organisms, leading to possible contamination or experimental artifacts.  
         [0003]     It is believed that certain pathologies are associated with bacteria or bacterial forms which are difficult to detect, and which may pass through nano-porous barriers. This leads to possible errors in diagnosis or causation, and which may lead to erroneous treatment and impede prevention.  
         [0004]     See, Hopert, Anne, Uphoff, Cord C., Wirth, Manfred, Hauser, Hansjorg, and Drexler, Hans G., “Specificity and sensitivity of Polymerase Chain Reaction (PCR) in comparison with other methods for the detection of  mycoplasma  contamination in cell lines”, J. Immunological Methods, 164(1993):91-100, expressly incorporated herein by reference.  
         [0005]     Kajander, E. O., et al., “Comparison of  Staphylococci  and novel Bacteria-Like Particles from blood”, Zbl. Bakt. Suppl. 26, 1994, expressly incorporated herein by reference.  
         [0006]     Akerman, Kari K., “Scanning Electron Microscopy of Nanobacteria—Novel Biofilm Producing Organisms in Blood”, Scanning Vol. 15, Suppl. III (1993), expressly incorporated herein by reference. www.newcastle.edu.au/discipline/biology/projects/hons_cpru.html, expressly incorporated herein by reference.  
         [0007]     Cifticioglu, Neva, et al., “Apoptotic effect of nanobacteria on cultured mammalian cells”, Mol. Biol. Cell. Suppl., Vol. 7 (1996):517a  
         [0008]     Cifticioglu, Neva, et al., “A new potential threat in antigen and antibody products: Nanobacteria”, Vaccines 97, Brown et al. Ed., Cold Spring Harbor Laboratory Press, New York, 1997, expressly incorporated herein by reference.  
         [0009]     Baseman, Joel B., et al., “ Mycoplasmas:  Sophisticated, Reemerging, and Burdened by their Notoriety”, EID Vol. 3, N o  1 www.cdc.gov/ncidod/EID/vol3no1/baseman.htm, expressly incorporated herein by reference.  
         [0010]     Relman, David A., “Detection and Identification of Previously Unrecognized Microbial Pathogens”, EID Vol. 4, N o  3 www.cdc.gov/ncidod/EID/vol4no3/relman.htm, expressly incorporated herein by reference.  
         [0011]     Mattman, Lida H., Cell Wall Deficient Forms-Stealth Pathogens, 2nd Ed., CRC Press (1993), expressly incorporated herein by reference.  
         [0012]     U.S. Pat. No. 5,688,646, expressly incorporated herein by reference, describes novel  mycoplasmas  which are prominent in patients who are thought to be suffering from AIDS. Devices are also provided for the in vitro detection of  mycoplasmas  in biological fluid by means of a reagent which is specific for the  mycoplasma  group without being specific for particular species within said group. Devices for testing  mycoplasma  sensitivity to antibiotics are also described.  
         [0013]     See the following, each of which and cited references is expressly incorporated herein by reference in their entirety:  
                                             Pat. No   Title                                6,562,611   FEN-1 endonucleases, mixtures and cleavage methods       6,558,902   Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of           macromolecules       6,555,357   FEN-1 endonuclease, mixtures and cleavage methods       6,555,338   NrdF from  Staphylococcus aureus         6,537,774   UPS (undecaprenyl diphosphate synthase       6,492,113   Detection of  Mycoplasma  genus and species in patients with chronic fatigue syndrome           and fibromyalgia       6,489,139   FabZ from  Staphylococcus aureus         6,489,110   EF-Tu mRNA as a marker for viability of bacteria       6,458,572   Phosphatidylglycerophosphate synthase from  Staphylococcus aureus         6,458,535   Detection of nucleic acids by multiple sequential invasive cleavages 02       6,448,037   PgsA       6,432,703   RATC from  Streptococcus pneumoniae         6,410,286   Asparaginyl tRNA synthetase from  Staphylococcus Aureus         6,399,343   inFB       6,372,424   Rapid detection and identification of pathogens       6,361,965   YfiI pseudouridine synthase       6,353,093   gidB       6,350,600   trmD       6,348,582   Prokaryotic polynucleotides polypeptides and their uses       6,348,328   Compounds       6,348,314   Invasive cleavage of nucleic acids       6,346,397   GyrA       6,340,564   yhxB       6,331,411   TopA       6,326,172   ytgP       6,312,932   Yfil pseudouridine synthase       6,309,866   6-phosphogluconate dehydrogenase       6,303,771   Pth       6,294,652   Response regulator       6,294,357   FabF from  Staphylococcus aureus         6,287,807   MurF       6,287,804   nrdG       6,277,595   FabZ       6,274,719   Gcp       6,274,361   pth       6,270,762   tdk       6,268,177   Isolated nucleic acid encoding nucleotide pyrophosphorylase       6,261,802   Ups (ugc)       6,261,769   Intergenic spacer target sequence for detecting and distinguishing  Chlamydial  species           or strains   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Histidine kinase       6,165,991   Sensor histidine kinase of  Streptococcus pneumoniae         6,165,764   Polynucleotides encoding tRNA methyl transferases from  Streptococcus pneumoniae         6,162,619   Sensor histidine kinase of  streptococcus pneumoniae         6,162,618   6-phosphogluconate dehydrogenase of  Streptococcus pneumoniae         6,156,537   Phospho-N-acetylmuramoyl-pentapeptide transferase of  Streptococcus pneumoniae         6,146,863     Staphylococcus aureus  3-hydroxyacyl-CoA dehydrogenase       6,146,846   Primosome protein a of  streptococcus pneumoniae         6,140,079   GidB       6,140,061   Response regulator       6,111,074   PyrH of  Streptococcus pneumoniae         6,110,723   Yfii pseudouridine synthase       6,110,685   infB       6,090,543   Cleavage of nucleic acids       6,060,294   Alanyl tRNA synthetase from  Staphylococcus aureus         6,001,567   Detection of nucleic acid sequences by invader-directed cleavage       5,994,111   Leucyl tRNA 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DNAs       6,312,903   Simulataneous detection, identification and differentiation of eubacterial taxa using a           hybridization assay       6,306,653   Detection and treatment of breast disease       6,300,091   Herbicide target genes and methods       6,300,072   PCR methods and materials for detecting  bartonella  species       6,287,779   Detection of fermentation-related microorganisms       6,268,142   Diagnostics and therapeutics for diseases associated with an IL-1 inflammatory           haplotype       6,261,773   Reagent for nucleic acid amplification and process for nucleic acid amplification       6,252,130   Production of somatic mosaicism in mammals using a recombinatorial substrate       6,251,607   PCR primers for the rapid and specific detection of  Salmonella typhimurium         6,248,519   Detection of fermentation-related microorganisms       6,221,582   Polynucleic acid sequences for use in the detection and differentiation of prokaryotic           organisms  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20030138772   Method of detecting and/or identifying adeno-associated virus (AAV) sequences and           isolating novel sequences identified thereby       20030135879   Methods and materials for making and using transgenic dicamba-degrading organisms       20030134343   Specific mucin expression as a marker for pancreatic cancer       20030134310   Cellular kinase targets and inhibitors, and methods for their use       20030134301   Identification and use of molecules implicated in pain       20030134295   Method for detection of pathogenic organisms       20030134293   Method for rapid and accurate identification of microorganisms       20030134275   Telomerase reverse transcriptase (TERT) genes       20030131377   DNA molecules from maize and methods of use thereof       20030131375   Plant regulatory sequences for selective control of gene expression       20030129746   Epitopes in viral envelope proteins and specific antibodies directed against these           epitopes: use for 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       SUMMARY OF THE INVENTION  
       [0014]     The present invention allows improved detection of such bacterial contaminants, including unconventional forms such as filtering forms (nanoforms), nanobacteria, and L-forms.  
         [0015]     Its principal applications are the: 
        Detection of very low levels of  mycoplasma  contamination of cell lines and biological fluids     Identification of latent bacterial infections in various pathologies     Detection of live forms passing through filters having a pore size of between 100 and 20 nm     Direct detection of RNA-containing subunits of bacteria.        
 
         [0020]     Different primers have been designed:  
         [0021]     MOLL primers have been designed initially to detect mollicutes ( mycoplasma ) species based on the conserved regions of the 16s ribosomal RNA gene. In fact they can also detect gram positive bacteria  
                                   1)   Moll Outer Primer (sense)   (AAYGGGTGAGTAACACGT),                   2)   Moll Outer Primer (antisense)   (CCCGAGAACGTATTCACCG)               3)   Moll Inner Primer (sense)   (CTACGGGAGGCAGCAGTA)               4)   Moll Inner Primer (antisense)   (GTATCTAATCCTRTTTGCTCCCCA)          
 
         [0022]     BACT primers will detect gram positive and gram negative bacteria. The sequence of MOLL primers is included in the degenerated sequence of the BACT primers.  
                               1)               Bact Outer Primer (antisense)   (CCCGRGAACGTATTCACSG),               2)       Bact Inner Primer (sense)   (CTACGGGAGGCWGCAGTRRGGAAT),               3)       Bact Inner Primer (antisense)   (WGGGTATCTAATCCTRTTTGMTCCCCW)          
 
         [0023]     The GNEG set of primers is specific of gram negative bacteria. It differs from the Moll 16 out S by a single nucleotide 
        1) Gneg Outer Primer (sense) (RAYGGGTGAGTAAYGYMT),        
 
         [0025]     The present invention therefore provides a method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first amplified genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.  
         [0026]     It is believed that the Nanoforms are a stable, low metabolic rate form of bacteria, which may be related to pathology, which have characteristic DNA which is generally undetectable by PCR or nested PCR. However, these organisms do have characteristic RNA, and therefore these can be detected by nested RT-PCR. Likewise, because these are now detectable according to the present invention, it is therefore possible to monitor and optimize treatments directed toward clearing these from infected subjects.  
         [0027]     It is believed that Nanoforms are involved in human pathology, and further that these low metabolic organisms are involved in a constellation of chronic human diseases. Further, it is believed that some of these Nanoforms may be subcellular, that is, incomplete, and therefore may require association with other Nanoform, or other organisms or cells, for replication or reconstitution as a complete DNA bacterial form. Preliminary evidence suggests that the genetic material within a single Nanoforms is insufficient to reconstitute the entirety of a related bacterial (DNA) form, and therefore that multiple Nanoforms may be required in order to be self-replicating for the complete organism.  
         [0028]     For example, multiple Nanofoms may infect a single cell, together constituting a complete genome for the associated DNA bacteria. Reverse transcriptase activity, for example, due to retroviruses, endogenous retroviral sequences, DNA pol I activity, etc., may be sufficiently active to generate the bacterial genome.  
         [0029]     These Nanoforms may be biologically associated with retroviruses, such as HIV, which would therefore increase their likelihood of replication, since they would then carry their own reverse transcriptase, and potentially account for replication of sub-cellular fragments. The retroviruses may be passengers within the Nanoforms, and the Nanoforms represent an infectious particle for the virus.  
         [0030]     The present invention reveals that the Nanoforms retain conserved sequences of 16S rRNA, and therefore may be targeted on this basis, for example by tetracycline analog antibiotics, especially administered over extended durations.  
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
     Material and Methods  
       [0000]     Oligonucleotide Primers.  
         [0031]     The oligonucleotide primers used were:  
                               1)               Moll Outer Primer (sense)   (AAYGGGTGAGTAACACGT),               2)       Gneg Outer Primer (sense)   (RAYGGGTGAGTAAYGYMT),               3)       Bact Outer Primer (antisense)   (CCCGRGAACGTATTCACSG),               4)       Bact Inner Primer (sense)   (CTACGGGAGGCWGCAGTRRGGAAT), and               5)       Bact Inner Primer (antisense)   (WGGGTATCTAATCCTRTTTGMTCCCCW)         where R = G or A, S = G or C, W = A or T, M = A or C, and Y = T or C.             
 
         [0032]     Expected lengths of amplicons are ˜1,200 bp and ˜450 bp with the outer and inner primers, respectively. The primers employed were formulated as equal amounts of each primer within the class identified by the sequence.  
         [0000]     Nucleic Acid Preparation and PCR/RT-PCR.  
         [0033]     Cell line supernatants (400 μl), human plasma (200-400 μl) and human peripheral blood mononuclear cells (PBMC, 3-10 millions cells) were lyzed with 10 mM Tris, pH 7.4, 10 mM EDTA, 150 mM NaCl, 0.4% SDS, and 10 μg Proteinase K at 60° C. for 1 h. Nucleic samples were extracted three times with one volume of phenol/chloroform and one time with chloroform and precipitated by addition of 1/10 volume of 3M sodium acetate and two volumes of ethanol at −60° C. for 1 h. Samples were centrifuged 30 min. and the nucleic acid pellets were washed with 70% cold ethanol and solubilized in 10 mM Tris-HCI, pH 8.0. These preparations were stored at −60° C.  
         [0034]     PCR reaction mix (50 μl) consisted of 5 mM MgCl2, 50 mM Tris, pH 8.0, 15 mM (NH4)2SO4, 10 mM B-Mercaptoethanol, 500 μM dATP, dCTP, dGTP, and DTTP, 0.025% BSA, 1 μM of each outer primer, 1 U Taq polymerase (Roche Molecular biochemicals, Laval, Canada), and 5-10 μl nucleic acid sample. For the first round PCR, the denaturation, annealing, and elongation temperatures and times used were 95° C. for 30 s, 42° C. for 30 s, and 78° C. for 2 m, respectively, for 42 cycles. After the final cycle, the products were kept at 78° C. for 10 m. One μl of the PCR product was subjected to a second round PCR with the set of inner primers. Denaturation, annealing, and elongation temperatures and times used were 95° C. for 30 s, 47° C. for 30 s, and 78° C. for 1 m, respectively, for 42 cycles, followed by a single incubation at 78° C. for 10 m. After the first and second round PCR, 10 μl of PCR product was analyzed by gel electrophoresis using 1.5% agarose, stained with ethidium bromide, visualized under ultraviolet light and photographed. Visible bands with appropriate size were cut and sequenced using the inner primers (DNA Landmarks, St-Jean sur le Richelieu, Canada). Sequence homology search was performed using the BLAST program of the NIH web site.  
         [0035]     Samples negative for the appropriate band by PCR were subjected to a first round RT-PCR followed by a second round PCR. RT-PCR reaction mix (50 μl) consisted of 5 mM MgCl2, 50 mM Tris, pH 8.0, 15 mM (NH4)2SO4, 10 mM B-Mercaptoethanol, 500 μM dATP, dCTP, dGTP, and DTTP, 0.025% BSA, 1 μM of each outer primer, and Titan enzyme mix (Roche Molecular biochemicals, Laval, Canada), and 5 μl nucleic acid sample. The reverse transcription step was performed at 42° C. for 30 m. The first and second rounds PCR were performed as described above.  
         [0036]     The precautions addressed elsewhere (Kwok and Higuchi, 1989) were followed to minimize the risk of false-positive results caused by the carry-over of previously amplified DNA. For example, extraction of nucleic acids and preparation of PCR/RT-PCR mix were performed under a sterile flow bench, only aliquoted reagents and filter tips were used, and negative controls were incorporated into each run.  
       Results  
       [0000]     All Samples:  
         [0037]     First round PCR/RT-PCR: no detection of expected amplicon (˜1,200 bp).  
         [0038]     Second round PCR (nested-PCR): all amplicon (˜450 bp) sequences related to bacterial 16S ribosomal RNA gene.  
         [0000]     Patients&#39; Lymphocytes (18 Samples):  
         [0039]     No 450 bp amplicon detected by nested-PCR from first round PCR.  
         [0040]     All samples positive (450 bp) by nested-PCR from RT-PCR.  
         [0041]     Therefore, bacteria are in an “RNA state”, and are referred to herein as “Nanoforms”.  
         [0000]     Samples of Patients&#39; Plasma:  
         [0042]     11 samples/12 positive for 450 bp amplicon by nested-PCR from first round PCR.  
       Discussion  
       [0043]     Cell wall deficient pathogenic microorganisms, which may be  mycoplasma,  so-called L-forms, or potentially other types, are difficult to detect. Therefore, their involvement in pathology may be vastly under-reported.  
         [0044]     It has been found, however, that these organisms have a well-conserved RNA sequences, such as the 16S rRNA, even when corresponding DNA or RNA is undetectable by a traditional polymerase chain reaction (PCR) or reverse transcriptase PCR (RT-PCR) method, which may be detected by nested RT-PCR amplification, using primers according to the present invention.  
         [0045]     The present invention therefore provides a sensitive and specific method for detecting bacterial forms, which may be called “Nanoforms”, even when traditional methods fail. This therefore allows diagnosis of pathogens previously unrecognized, and monitoring of treatment thereof.