PATENT ABSTRACT
The present invention relates to nucleic acid molecules which allow the identification of bacteria or groups of bacteria. For detection, the region of the bacterial genome containing the 23 S/5 S rRNA is used as the target sequence for the bacterial detection.

PATENT DESCRIPTION
The present invention relates to nucleic acid molecules which allow the identification of bacteria or groups of bacteria. 
     Bacteria are an ubiquitous component of the human environment. But they cause problems so frequently, as agents of food spoilage or pathogens, that effective, rapid, and reliable diagnosis is of great importance. 
     The most important microorganisms which cause food spoilage are  Clostridium botulinum , the cause of botulism;  Campylobacter jejuni; Clostridium perfringens; Cryptosporidium parvum , enteropathogenic strains of  Escherichia coli; Shigella; Listeria monocytogenes; Salmonella species; Staphylococcus aureus; Vibrio vulnificus ; and  Yersinia enterolytica . The General Accounting Office (GAO) reported in 1996 that from 6.5 to 81 million cases of food poisoning occur in the USA every year. The US Food and Drug Administration (FDA) estimates that 2–3% of all food poisonings lead to chronic secondary diseases. It is also estimated that 2–4 million cases of sickness in the US are caused by more than 2000 strains of  Salmonella . Those horrifying statistics could be extended to other food spoilage organisms. Food poisonings do not just cause human suffering, though, with death in extreme cases, but also substantial economic damage, which is estimated at 5.6–9.4 billion dollars for the US in 1991, for instance. 
     It is generally known that microorganisms, as agents of infection, present great danger. Their potential can hardly be estimated. For instance, the World Health Report from the WHO indicates statistical orders of magnitude. In 1998, for instance, pathogens, including parasites, were responsible for 9.8 million deaths (not counting prenatal or postnatal infections). That amounts to 18.2% of all deaths due to disease. The dangerous pathogens cannot be summarized as well as the food spoilage organisms, as they are recruited from many phylogenetic branches of the Eubacteria. There is a particularly great “infectious potential” in the Enterobacteria family, in particular. 
     In combating bacteria pathogenic for humans, identification of the microbes causing a disease or a pathologic symptom is a significant step. Often the proper medical measures can be applied only after the identification. Furthermore, detection methods for bacteria which work well could also be used as preventive tools in food quality assurance. 
     Classical detection of bacteria consists of microbiological identification, which usually involves isolation on selective media containing agar. This procedure has two significant disadvantages, however. First, the detection is often not reliable or specific. Second, many bacteria require a growth period of at least 18 hours for isolation as colonies. In many cases, a secondary isolation or a secondary detection are also necessary. Everything considered, diagnosis times up to a week are not unusual. In addition to that, there are also pathogenic microbes which cannot be cultured (J. J. Byrd et al., 1991, Appl. Environ. Microbiol. 57, 875–878). In a time of rapid means of transport and global trade in goods, though, rapid diagnostic methods which in the optimal case should not take longer than 24 hours, are essential to prevent the spread of pathogens or world-wide food poisonings from just a single local source. 
     Various procedures have been developed in recent years to meet modern requirements. They are intended to provide rapid and reliable routine identification of microbes. For example, immunologic methods utilize the specific binding of monoclonal or polyclonal antibodies to bacterial surface antigens. Such procedures are used particularly for serotyping for  Salmonella , for instance. In general, to be sure, detection by ELISA is relatively rapid, but it requires processing and isolation of the specific antigens, and that can have many problems. Bacterial detection methods utilizing DNA probes have proven to be particularly capable because they are very sensitive, relatively specific, and can be used to detect microorganisms in a total experimental period of 2–3 days. 
     BACKGROUND OF THE INVENTION 
     The invention consists in providing specific DNA sequences and selecting DNA regions which are particularly suitable for detecting bacteria. Thus this application is based on the identification of organisms by their genetic information. Using deviations of as little as a single component in the nucleotide sequence in certain DNA regions it is already possible to differentiate species. 
     Historically considered, ribosomal RNA genes have already been used for phylogenetic classification of organisms. Comparisons of sequences of the 5 S and 16 S ribosomal genes in different bacteria have led to significant corrections in assignments of relatedness and to discovery of the kingdom of the Archaebacteria. Because of its size and the corresponding high sequencing effort, 23 S RNA has only in recent years been used for systematic classifications. 
     Direct sequencing of genes of microorganisms to be identified was too expensive and time-consuming in practical use. In the 1980s, therefore, specific nucleotide probes were used to detect bacteria. While those can show very good specificity, the detection limit is often too low. The probe technology was substantially improved by combination with amplification techniques, which reproduce the nucleotide sequence to be detected and thus substantially increase the sensitivity of detection. In an extreme case, it is possible to detect a single isolated genome. In practice, losses occur in isolation of DNA, increasing the detection limit to about 10 2  to 10 4  cells. 
     On the basis of fundamental research, DNA probes from the 5 S, 16 S and 23 S genes were utilized for practical applications. For instance, one should note these patents: Nietupski et al. (U.S. Pat. No. 5,147,778) for detection of  Salmonella ; Mann and Wood (U.S. Pat. No. 6,554,144) for detection of  Yersinia  species; Leong (EP 04 79 117 A1) for detection of various Gram negative and Gram positive bacteria; Carico et al. (EP 1 33 671 B1) for detection of various enterobacterial species; Shah et al. (EP 03 39 783 B1) for detection of  Yersinia enterolytica ; Carrico (EP 01 63 220 B1) for detection of  Escherichia coli ; Hogan et al. (WO 88/03957) for detection of species of  Enterobacteria, Mycobacterium, Mycoplasma  and  Legionella ; Leiser et al. (WO 97/41253) for detection of various microorganisms; Grosz and Jensen (WO 95/33854) for detection of  Salmonella enterica ; Stackebrandt and Curiaie (EP 03 14 294 A2) for detection of  Listeria monocytogenes ; Wolff et al. (EP 04 08 077 A2), Hogan and Hammond (U.S. Pat. No. 5,681,698) for detection of  Mycobacterium kansasii ; Hogan et al. (U.S. Pat. No. 5,679,520) for detection of various bacteria; Kohne (U.S. Pat. No. 5,567,587) particularly for detection of bacterial RNA; Kohne (U.S. Pat. No. 5,714,324) for detection of various bacteria; Pelletier (WO 94/28174) for detection of Legionella; and Kohne (U.S. Pat. No. 5,601,984) for detection of various bacteria. Most of the patents relate to the sequence of the 16 S rDNA gene, and many also relate to the 23 S rDNA. 
     It appeared, though, that the latter genes are not suitable for many differentiation operations in practical use because they are too strongly conserved. Closely related microorganisms in particular cannot be differentiated. On the other hand, the 5 S rDNA gene is generally too variable and its differentiation potential is too low for practical use, even though it was initially used for phylogenetic studies in basic research because of its small size. 
     As the 5 S, 16 S and 23 S rDNA genes have many disadvantages as diagnostic aids, DNA regions which could be used for identification of all eubacteria were sought. Such a DNA region should have very variable and, at the same time, strongly conserved sequences. Then the variable regions would be useful to differentiate closely related species, such as strains and species. The conserved sequences would be used to detect more distantly related bacteria or higher taxonomic units. 
     In the very recent past, the 16 S–23 S transcribed spacer has been discussed in the literature in the context of extensive studies on ribosomal operons. Their applicability in systematic bacteriology has been questioned, though. For example, Nagpal et al. (J. Microbiol. Meth. 33, 1998, p. 212) considered the utility of these spacers very critically: A major problem with this transcribed rDNA spacer is that it frequently contains tRNA insertions. Such insertions represent dramatic changes in the sequences, and do not necessarily have a relation to phylogenetic separations. However, they have been used in the past to utilize the length polymorphism which they cause as a phylogenetic characteristic (Jensen et al. 1993, Appl. Envir. Microb. 59, 945–952; Jensen, WO 93/11264; Kur et al. 1995, Acta Microb. Pol. 44, 111–117). 
     The transcribed spacer between the 23 S and 5 S rDNA is an alternative target sequence for identification of bacteria. For instance, Zhu et al. (J. Appl. Bacteriol. 80, 1996, 244–251) published detection of  Salmonella typhi  using this diagnostic DNA region. However, the general utility of this spacer for detecting other bacteria cannot be derived from that work. There are very many examples which indicate that a DNA region is suitable only for identifying one or a few species of bacteria. Individual patents imply a potential but very limited applicability of the 23 S–5 S transcribed DNA region for bacterial diagnosis. Those all have in common that their applicability is limited to just a single bacterial species, specifically, to detection of  Legionella  (Heidrich et al., EP 07 39 988 A1),  Pseudomonas aeruginosa  (Berghof et al., DE 197 39 611 A1) and  Staphylococcus aureus  (Berghof et al., WO 99/05159). 
     The technical problem underlying the present invention consists in providing materials and processes which allow to detect any desired bacterium (preferably from the Enterobacteria group) in a material being examined. 
     This problem is solved according to the invention by a nucleic acid molecule as a probe and/or a primer for detection of bacteria, selected from
         a) a nucleic acid comprising at least one sequence with any of the SEQ ID NOs: 1 to 530 and/or a sequence from position 2667 to 2720, 2727 to 2776, 2777 to 2801, 2801 to 2832, 2857 to 2896, 2907 to 2931, 2983 to 2999, and/or 3000 to 3032 according to SEQ ID NO: 1; or nucleic acids homologous with them;   b) a nucleic acid which hybridizes specifically with a nucleic acid according to a);   c) a nucleic acid which exhibits 70%, and preferably at least 90%, identity with a nucleic acid according to a) or b);   d) a nucleic acid which is complementary to a nucleic acid according to any of a) to c);   and/or   combinations of the nucleic acids according to any of a) to d), except for the SEQ ID NO:1.       

     Further claims concern preferred embodiments. 
     In one particularly preferred embodiment, the presence of Enterobacteria in a sample being analyzed is shown by the analysis sample being brought into contact with a probe which detects the presence of a nucleic acid from the 23 S/5 S rDNA genome segment of the Enterobacteria. 
     The sequence specified as NO: 1 in claim  1  is derived from  E. coli . Homologous DNA sequences are those derived from bacteria other than the  E. coli  sequence shown, but in which the genome segment from the other bacteria corresponds to the sequence based on SEQ ID NO:1. For more details, we refer to the definition of homologous DNA sequences, below. 
     The nucleic acid molecule according to the invention comprises preferably at least 10 nucleotides, and especially preferably at least 14 nucleotides. Nucleic acid molecules of these lengths are used preferably as primers, while nucleic acids used as probes preferably comprise at least 50 nucleotides. 
     In another preferred embodiment, nucleotides of the probe or the primer can be replaced by modified nucleotides containing, for instance, attached groups which ultimately are used for a detection reaction. Particularly preferred derivatizations are specified in claim  4 . 
     In another preferred embodiment, combinations of the specified nucleic acid molecules are used. Selecting the particular combination of nucleic acid molecules allows adjustment of the selectivity of the detection reaction. In doing so, selection of the primer combinations and/or probe combinations can establish the conditions of the detection reactions so that they either demonstrate generally the presence of bacteria in a sample, or specifically indicate the presence of a certain bacterial species. 
     A kit according to the invention contains at least one nucleic acid according to the invention together with the other usual reagents used for nucleic acid detection. They include, among others, suitable buffers and detection agents such as enzymes with which, for example, biotinylated nucleic acid hybrids which are formed can be detected. 
     In another preferred embodiment, called Consensus PCR here, the process is carried out according to claim  8 . First, a nucleic acid fragment is amplified by use of conserved primers (those hybridize to nucleic acids of different bacterial taxonomic units). Then more specific nucleic acid segments are detected by use of other more specific nucleic acids (these hybridize with only a few taxonomic units or only with a certain species). The latter allow then a conclusion about the presence of a particular genus, type or species in the sample being analyzed. 
     Various established detection procedures can be employed to detect nucleic acids in the process used. They include Southern Blot techniques, PCR techniques, LCR techniques, etc. 
     In one broad study, transcribed spacer between 23 S and 5 S rDNA was examined for its general usefulness as a diagnostic target molecule. For this purpose, genomic DNA from very many bacterial strains was isolated, purified, cloned into a vector, sequenced, and finally evaluated in an extensive sequence comparison. Surprisingly, this sequence segment was suitable for identification of almost all bacterial species. With the encouragement of that finding, the analyses were extended to the adjacent regions of the spacer. All in all, DNA fragments from all bacterial classes or smaller phylogenetic units were examined. They have lengths of 400–750 base pairs and include the end, i.e., the last 330–430 nucleotides (depending on the species) of the 23 S rDNA gene, the transcribed spacer, and the complete 5 S rDNA gene. The total size of the fragments is 400–750 base pairs. The experiments showed that the 23 S rDNA gene and the 5 S rDNA gene are adjacent in almost all bacterial species. This information is an important prerequisite for use and applicability of this invention. 
     This invention is particularly based on the fact that a DNA region which can contain significant portions of at least two adjacent genes is selected for detection of microorganisms. In practice, the usefulness of the region is determined particularly by its phylogenetic variability. There can be quite contrary requirements, depending on whether distantly related bacteria, taxonomic units, or strains of a species are to be detected. Now the frequency of occurrence of both variable and conserved regions is greater for two genes than for one, as the example of the 23 S–5 S tandem shows. Thus the use of two adjacent genes, including the variable intercalated sequences is a substantial advantage. 
     It was also found that the end of the 23 S rDNA gene, the 5 S rDNA gene, and the transcribed spacer between them contain nucleotide sequences which cover a wide range from very variable to very conserved. A fine analysis of this region provided further very interesting conclusions about the differentiation potential of various phylogenetic bacterial units ( FIG. 2 , Table 6). Nearly all taxonomic units can be detected and/or differentiated by using subregions. More or less variable regions are shown in  FIG. 2  with the sections 1–9, while the strongly conserved regions are intercalated between and adjacent to them. The latter are thus particularly suitable for detecting higher taxonomic units, such as the whole Eubacteria or classes or divisions of them. 
     The phylogenetic dendrogram in  FIG. 1  provides another indication of the usefulness of the region. It can be seen that the 23 S rDNA–5 S rDNA region allows very good differentiation with respect to coarse classification, as members of the Proteobacteria are assigned to 1–2 groups, while the Firmicutes are separated. Furthermore, the lengths of the branches, even for closely related species, indicates that they can be distinguished well from each other. Here a phylogenetically correct assignment of close relatives in the dendrogram is quite undesirable, because then they would lie in a closely connected coherent group and perhaps could not be distinguished as easily from one another. 
    
    
     
       DETAILED DESCRIPTION OF THE FIGURES 
         FIG. 1 : Phylogenetic dendrogram of some bacteria detected in this work. It can be seen that the Proteobacteria and the Firmicutes form branches which can be separated. 
         FIG. 2 : Schematic representation of the ribosomal region described herein comprising the terminal region of the 23 S rDNA, the transcribed spacer, and the 5 S rDNA. This region, or parts of it, is used to detect bacteria. Table 6 shows a detailed characterization of individual domains. 
         FIGS. 3–7 : Detection of enterobacteria by PCR. The figures show gels stained with ethidium bromide. The presence of bands is characteristic of the presence of Enterobacteria. The upper halves of the figures show positive findings, while the lower halves show the negative controls. Table 7 summarizes the use of the primer. A mixture of Bgl 1 and Hinf 1 of restriction-digested BR328 plasmid DNA (Boehringer Mannheim) was used as the DNA size standard. The DNA size markers include the restriction fragment sizes 154, 220, 234, 298, 394, 453, 517, 653, 1033, 1230, 1766 and 2176 base pairs. 
         FIG. 8 : Plan of a consensus PCR. Conserved primers are arranged peripherally, and less-conserved primers are nested internally. In a first step, consensus PCR allows amplification of DNA with high taxonomic breadth, in the extreme case of all bacterial species. In the subsequent steps, there can be further rounds of amplification. They may be performed in separate vessels, with primers specific for smaller taxonomic units. In the final step, probes can be used which likewise contribute to the specificity of the detection and which can also aid observation of the detection, such as with dyes. Here, and in this figure, the following nomenclature is used: Primer A: the most conserved primers, and the ones with the most peripheral positions in the detection system; Primer [A, B, C . . . ]: the sequence of primers in the nesting as shown above; Primer [capital letter]1: forward primer; Primer [capital letter]2: reverse primer; Primer [capital letter][number][lower-case letter]: the lower-case letters characterize similar primers, or primers which hybridize at homologous or comparable positions within a target DNA. The probe is preferably in the central, highly variable, region if species or strains are to be detected. 
     
    
    
     EXAMPLE 1 
     Detection of the Enterobacteriaceae Family 
     Genomic DNA was isolated, using standard procedures which are themselves known, from pure cultures of the bacteria listed in Table 1. Quantities of about 1 to 100 ng from each of these preparations were used in PCRs. The reaction solution had the following composition: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 genomic DNA 
                 1 
                 μl 
               
               
                   
                 H 2 O 
                 19.8 
                 μl 
               
               
                   
                 Buffer (10×) *1   
                 2.5 
                 μl 
               
               
                   
                 dNTP (10 mM) *2   
                 0.25 
                 μl 
               
               
                   
                 forward primer (10 μM) *3   
                 0.20 
                 μl 
               
               
                   
                 reverse primer (10 μM) *3   
                 0.20 
                 μl 
               
               
                   
                 MgCl 2   
                 0.75 
                 μl 
               
               
                   
                 Taq polymerase (5 U/μl) *1   
                 0.3 
                 μl 
               
               
                   
                   
               
               
                   
                   *1 Buffer and enzyme from Biomaster or any other source. 
               
               
                   
                   *2 Nucleotides from Boehringer Mannheim or any other source. 
               
               
                   
                   *3 Equimolar quantities of primers. 
               
               
                   
                 In the case of mixtures, each forward and reverse primer has a total final concentration of 10 μM. 
               
             
          
         
       
     
     The PCR was done in a Perkin Elmer 9600 Thermocycler with the thermal profile shown below: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 initial denaturation 
                 95° C. 
                  5 minutes 
               
               
                   
                 amplification (35 cycles) 
                 92° C. 
                  1 minute 
               
               
                   
                   
                 62° C. 
                  1 minute 
               
               
                   
                   
                 72° C. 
                 30 seconds 
               
               
                   
                 final synthesis 
                 72° C. 
                  5 minutes 
               
               
                   
                   
               
             
          
         
       
     
     The species listed in Table 1 were tested for identification of the Enterobacteriaceae family. The primer combinations used and the primer-specific parameters are listed in Table 7. When more than one forward or reverse primer is listed in Table 7, it indicates use of that mixture. 
     The result of the PCR was analyzed by agarose gel electrophoresis and staining with ethidium bromide. The presence of PCR products indicates the presence of enterobacteria. 
     The synthesized PCR products are mostly of sizes on the order of 400 to 750 base pairs. Many bands can occur throughout, because ribosomal alleles are heterogeneous in many bacterial species. Table 1 shows the results obtained. They show that the enterobacteria are completely delimited from representatives of other taxa. 
     EXAMPLE 2 
     Detection of a Bacterial Species, with  Pantoea Dispersa  as an Example 
     Genomic DNA can be isolated from pure cultures of bacteria by standard procedures which are themselves known. Quantities of about 1 to 100 ng each from these preparations can be used in a PCR. The reaction solution can then have the following composition: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 genomic DNA 
                 1 
                 μl 
               
               
                   
                 H 2 O 
                 19.8 
                 μl 
               
               
                   
                 Buffer (10×) *1   
                 2.5 
                 μl 
               
               
                   
                 dNTP (10 mM) *2   
                 0.25 
                 μl 
               
               
                   
                 forward primer A (10 μM) *3   
                 0.20 
                 μl 
               
               
                   
                 reverse primer (10 μM) *3   
                 0.20 
                 μl 
               
               
                   
                 MgCl 2   
                 0.75 
                 μl 
               
               
                   
                 Taq polymerase (5 U/μl) *1   
                 0.3 
                 μl 
               
               
                   
                   
               
               
                   
                   *1 Buffer and enzyme from Biomaster. 
               
               
                   
                   *2 Nucleotides from Boehringer Mannheim or any other source. 
               
               
                   
                   *3 Equimolar quantities of primers. 
               
               
                   
                 In the case of mixtures, each forward and reverse primer has a total final concentration of 10 μM. 
               
             
          
         
       
     
     The primer combinations SEQ ID 2+primer x1, SEQ ID (3–6)+primer x1, or the sequence complementary to primer x1+the sequence complementary to SEQ ID 147 can be used to detect  Pantoea dispersa . Here primer x1 is equivalent to the nucleotide CGTTGCCCCGCTCGCGCCGCTCAGTCAC. Primer x1 is a partial sequence from SEQ ID 108. 
     The PCR can be done in a Perkin Elmer Thermocycler with the thermoprofile shown below: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 initial denaturation 
                 95° C. 
                  5 minutes 
               
               
                   
                 amplification (35 cycles) 
                 92° C. 
                  1 minute 
               
               
                   
                   
                 62° C. 
                  1 minute 
               
               
                   
                   
                 72° C. 
                 20 seconds 
               
               
                   
                 final synthesis 
                 72° C. 
                  5 minutes 
               
               
                   
                   
               
             
          
         
       
     
     The result of the PCR can be made visible by agarose gel electrophoresis and staining with ethidium bromide. The synthesized PCR products have sizes on the order of 370, 320 and 70 base pairs. The absence of amplificates indicates absence of genomic DNA from  Pantoea dispersa . This experimental system can give the results summarized in Table 2. 
     EXAMPLE 3 
     Use of a Consensus PCR in Chip Technology 
     3a) Principle of Consensus PCR 
     In a consensus PCR, such as is shown schematically in  FIG. 8 , at least two “consensus primers” (A1, A2) are used, which can detect DNA from at least two taxonomic units. Those units can be strains, species, or even higher taxonomic units such as kingdoms or classes. In the detection system, the amplified taxonomic units are subsequently differentiated, in at least a second detection step, using another PCR and/or with probes. The PCR primers (B1, B2) of the second, or subsequent, amplification step are each chosen so that they are within the amplification product and have the potential to detect a specific taxonomic unit. By use of more primers (C, D, E . . . ), a pool of many taxonomic units can, if necessary, be narrowed down simultaneously. Furthermore, the detection potential can be extended to more taxonomic units in a multiplex mixture (such as A1a, A1b, A1c . . . ). The latter case exists if individual nucleotides in a primer differ or if the primers are completely different. The nomenclature of the consensus primers can also be found in the legend for  FIG. 8 . 
     Amplification products can be identified by means of the primers. The detection is positive if the primers recognize the target DNA and successfully amplify it. In addition probes can provide a specific detection. They hybridize specifically to the amplified DNA and allow a certain DNA sequence to be detected by direct or indirect coupling to dyes. Everything considered, probes can be used in many technical embodiments known to those skilled in the art. For example, there are Southern Blotting, the lightcycler technology with fluorescent probes, or the chip technology, in which arbitrarily many probes are arranged in a microarray. 
     It is particularly advantageous for success of a consensus PCR that the primers become increasingly specific in the order A, B, C . . . . That can be assured by selection of the DNA target region as shown in  FIG. 2 . 
     Consensus PCR has the advantage that it allows simultaneous detection of more than two taxonomic units from just a single nucleic acid sample, which can be correspondingly small. The number of detectable microorganisms can be increased in various ways. For instance, the detection potential of a consensus system increases with the number of primer species A, B, C, or A1a, A1b, A1c, . . . as they are defined in  FIG. 8 . In addition, a PCR solution can, after an initial process with a primer pair A1, A2, be separated and amplified in separate solutions with additional primer pairs B1a+B2a on the one hand and B1b+B2b on the other hand. Finally, the identity of PCR amplificates can be determined by hybridizing with probes. 
     3b) Example of Detection a Group of Genera of the Enterobacteria. 
     Genomic DNA can be isolated from pure cultures of bacteria by standard procedures which are themselves known. Quantities of about 1 to 100 ng each from these preparations can be used in a PCR. The reaction solution can have the following composition: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 genomic DNA 
                 1 
                 μl 
               
               
                   
                 H 2 O 
                 19.8 
                 μl 
               
               
                   
                 Buffer (10×)* 1   
                 2.5 
                 μl 
               
               
                   
                 dNTP (10 mM)* 2   
                 0.25 
                 μl 
               
               
                   
                 forward primer A (10 μM)* 3   
                 0.20 
                 μl 
               
               
                   
                 reverse primer (10 μM)* 3   
                 0.20 
                 μl 
               
               
                   
                 MgCl 2   
                 0.75 
                 μl 
               
               
                   
                 Taq polymerase (5 U/μl)* 1   
                 0.3 
                 μl 
               
               
                   
                   
               
               
                   
                 * 1 Buffer and enzyme from Biomaster. 
               
               
                   
                 * 2 Nucleotides from Boehringer Mannheim or any other source. 
               
               
                   
                 * 3 Equimolar quantities of primers. 
               
               
                   
                 In the case of mixtures, each forward and reverse primer has a total final concentration of 10 μM. 
               
             
          
         
       
     
     As chip technology generally uses very small reaction volumes, the reaction solution shown above can be made smaller with the concentrations remaining constant. It may be necessary to adjust the PCR cycle times. A ribosomal DNA fragment can be amplified initially for consensus PCR. That process can be specific for larger taxonomic units, as described in Example 1, with use of the primers described there. Alternatively, a ribosomal DNA fragment from all bacteria can be amplified. For instance, use of the primer combination SEQ ID 211+SEQ ID 212 provides ribosomal DNA of a very broad taxonomic spectrum of bacteria. 
     The amplified DNA is denatured by standard procedures, thus being converted into single-strand DNA. This form is able to bind to a DNA, RNA, or PNA probe. Then the hybridization of the amplificate is detected with the probe, depending on the design of the chip. Alternatively, detection can be done with an ELISA. The composition of the probe is such that it provides the specificity to meet the requirements. Accordingly, strains, genera, or larger taxonomic units can be detected. 
     Table 3 shows an example of detection of a group of genera of the family of the enterobacteria using the probe GTTCCGAGATTGGTT as a subsequence of SEQ ID 164. Such a group detection is particularly practical in chip technology if various group detections intersect with each other. Then an individual species, or groups of species, such as those important for food examinations, can be detected in the intersection. 
     3c) Use of Consensus PCR to Detect all Bacteria 
     To detect all bacteria, strongly conserved consensus primers are used in a first round of amplification. Suitable for selecting sequences are regions which are peripheral in the ribosomal segment, as shown in  FIG. 2 , are. They are consequently homologous to the regions of SEQ ID 1 beginning at position 2571 or ending at position 3112. From this region, for example, the primers SEQ ID 211 (as primer Ala, for instance) and SEQ ID 212 (as primer A2A, for instance) are particularly suitable for general amplification. Other primers (A1b, A1c, . . . , or A2b, A2c . . . ) which cover an arbitrarily large taxonomic range of the Eubacteria in a multiplex PCR can also be derived easily. In this nomenclature, primers A1 and A2 are primer pairs; B and C . . . are nested primers; and A1a and A1b are homologous or similar primers. 
     An initial differentiation can be accomplished by using nested primers (B, C, D . . . ). That can also be supported by dividing the primary PCR solution so that one primer pair B or C or D, etc., is used in each separate PCR solution. This nesting is particularly advantageous because the ribosomal region as shown in  FIG. 8  increases in variability from the outside to the inside, as is also described in Table 6. 
     Then it is preferable to use probes for final differentiation and identification. For instance, if species or strains are to be detected, then the probe should hybridize centrally in region 7 as shown in  FIG. 2 . 
     Table 8 presents many polynucleotides for detection of genera and species or strains in a consensus PCR. Use of primer number 1 from Table 8 has already been described extensively in Example 1. 
     The properties of the polynucleotides follow their characterization from Table 6 or  FIG. 2 . That means that primer A1 can be assigned to region 1 of Table 6 or  FIG. 2 ; primer A2 can be assigned to region 2 . . . ; primer B2 can be assigned to region 8, and primer A2 to region 9. According to this concept, primers A1–G1 from Table 8 can be used as forward primers, while primers B2 and A2 can be used as reverse primers. For that purpose, the sequences for the two latter primer types must be converted (Exception No. 1, Table 8). The “H1 primers” in particular can be used as genus-specific or species-specific probes. 
     The plan for a consensus PCR described here is not absolutely necessary for successful detection. In principle, the polynucleotides listed in Table 8 can be used in any arbitrary combination. In practice, one must first decide which bacteria are to be excluded from the detection as “undesired”. Then a simpler PCR version that differs from the plan shown can be selected, depending on the objective. The simplest form of consensus PCR, then, consists of just two primers corresponding to the sequences from Table 8, or sequences complementary to them. 
     Many of the conserved primers listed in Table 8 have the potential to detect the DNA of higher taxonomic units, such as classes, phyla, or families. As can be seen from Table 6, that applies particularly to the peripheral primer A or homologous sequences of SEQ ID 211+SEQ ID 212. Table 8 shows a broader potential for detecting one or more genera or species, particularly due to the redundant enumeration of the sequences. If only one sequence is explicitly listed for a genus, then two primers from that sequence can be selected for detection. It is also possible to select general primers, such as primer A of related genera, for the bacterial class of concern, and to sketch out a specific sequence, such as “primer h1” for a probe. As long as the sequences are very long, nucleotide fragments at least 15 bases long can be selected from them. 
     3d) Design of a Consensus PCR for Chip Technology 
     The actual design of a consensus PCR is determined essentially by the expected number of taxonomic units to be detected. As consensus PCR in its most complex form is also a multiplex PCR, only a limited number of bacteria can be determined in one reaction solution. Experience shows that this number is less than 20. For that reason, it can be advantageous to do different PCR solutions with the same probe and different primers A, B, etc. (nomenclature as shown in  FIG. 8 ). 
     First, bacteria from natural samples are enriched, or genomic DNA is isolated directly from them by standard procedures which are themselves known. Quantities of about 1 to 100 ng each from these preparations can be used in a PCR. The reaction solution can then have the following composition: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 genomic DNA 
                 1 
                 μl 
               
               
                   
                 H 2 O 
                 19.8 
                 μl 
               
               
                   
                 Buffer (10×)* 1   
                 2.5 
                 μl 
               
               
                   
                 dNTP (10 mM)* 2   
                 0.25 
                 μl 
               
               
                   
                 forward primer A (10 μM)* 3   
                 0.20 
                 μl 
               
               
                   
                 reverse primer (10 μM)* 3   
                 0.20 
                 μl 
               
               
                   
                 MgCl 2   
                 0.75 
                 μl 
               
               
                   
                 Taq polymerase (5 U/μl)* 1   
                 0.3 
                 μl 
               
               
                   
                   
               
               
                   
                 * 1 Buffer and enzyme from Biomaster. 
               
               
                   
                 * 2 Nucleotides from Boehringer Mannheim or any other source. 
               
               
                   
                 * 3 Equimolar quantities of primers. In the case of mixtures, each forward and reverse primer has a total final concentration of 10 μM. For example, primers can be designed and combined as described in 3c. 
               
             
          
         
       
     
     As very small reaction volumes are generally used in chip technology, the reaction solution above can be reduced in volume with the concentrations kept constant. Adjustment of the PCR cycle times may be necessary. 
     After the amplification rounds, the DNA is combined. Probes, which, in one specific embodiment, can be selected from the column “Primer H1” of Table 8 are immobilized on a chip. Technological procedures for that are known to those skilled in the art. The combined DNA is diluted 1:1 with denaturation buffer (Example 4) and incubated for one hour at room temperature. Then ten times that volume of hybridization buffer (Example 4) is added and the solution is slowly passed over the chip, i.e., the surface with probes adhering to it, at 37–60° C. After this procedure, the chip surface is washed three times for at least 2 minutes with wash buffer (Example 4) at 37–60° C. Then the detection can be done. Primers coupled to a fluorescent dye can be used for that. The fluorescence can be detected with a detector such as a CCD camera. However, there are various alternative possibilities for detection. For instance, it is also possible to follow and quantify the bonding of the single-stranded amplification products to the probes by surface plasmon resonance (SPR) spectroscopy. The latter method has the advantage that no dye need be used for detection. If SPR is used, it should be designed so that detection occurs simultaneously on the regions of the surface which have the same probes. A particularly advantageous embodiment has many (i.e., more than 100 or 1000) separate detection surfaces arranged on the chip. An increase in the SPR signal, caused by the nucleic acid hybridization on these surfaces, is a positive result. The primers listed in Table 8 can be used in this manner to detect the corresponding bacteria; or, in principle, to detect, and if required to quantify, all bacteria. 
     EXAMPLE 4 
     Detection of Microorganisms with Probes 
     Probes, being polynucleotides, i.e., DNA, RNA, PNA, or a similar embodiment known to those skilled in the art, are basically suitable for carrying out concentration and detection of DNA or RNA. They occur as single-stranded molecules, or they are converted to the single-stranded form by denaturation, such as by heating or by sodium hydroxide, according to published standard procedures. 
     To detect microorganisms, the DNA or RNA must be isolated from them and perhaps purified. Various measures can provide high efficiency in the nucleic acid yield:
     1) The microorganisms can be concentrated by physical methods, such as with antibodies coupled to magnetic particles, or by centrifuging.   2) The DNA or RNA from the microorganisms can be amplified in a PCR or comparable amplification reaction.   3) The DNA or RNA of the microorganisms, possibly amplified, is concentrated with commercially available material in the course of purification.   

     Improvement in the efficiency of nucleic acid yields, particularly through amplification, can itself contribute significantly to the specificity of bacterial detection. 
     This is followed by an incubation step, in which the probes form a hybrid molecule with the nucleic acids to be detected (if the microorganisms to be detected were present). The hybrid molecules are formed under controlled conditions. Then washing steps with buffers follow under conditions (pH, temperature, ionic strength) which allow specific hybridization of nucleic acids while less specific and undesired hybrid molecules dissociate. 
     Finally the hybrid molecules are detected. There are numerous procedures for detection, which are known in detail to those skilled in the art. Dyes, possibly fluorescent dyes, are used, which are coupled directly or indirectly to the probes or to the DNA being detected, or are incorporated into them. In particular, that can also happen in chip technology or in lightcycler technology. There are also other physical procedures, such as attenuated total reflection of light at interfaces with two different densities, which can be used in detection of hybrid molecules. 
     Evaluation of the detection can be done in various ways. In an “all or nothing” detection, the hybrid molecule can be detected only if the microorganism being sought were present. That is, if the previously mentioned amplification reaction with the nucleic acids of the microorganisms did not cause any multiplication of the amino acids, then no hybrid molecules will be detectable. However, if “undesired” nucleic acids were amplified, or if they had been present in large quantity, those nucleic acids can be excluded by the stringency conditions in hybridization. Also, quantification of the hybrid molecules allows fine tuning of the specificity of the detection, by establishing a limit for positive detection.
         All the nucleic acids specified in this patent are basically usable as probes. In particular, Table 3 lists an extract of possible probes. The nucleic acids provide detection of the genera specified in the table, and distinction from all other genera of the Eubacteria.       

     Examples are presented in the following of how the DNA regions specified for this purpose can be used as probes to detect microorganisms. An ELISA detection procedure is used in this example. In that procedure, nucleic acids are detected by an enzymatic reaction which proceeds in microtiter plates. 
     In this example, the DNA is first amplified in a PCR reaction. That reaction employs primers coupled with digoxigenin. Then a microtiter plate coated with streptavidin is loaded with a biotin-labeled probe, so that the probes couple to the plate surface. The PCR amplificates, denatured by base, hybridize with the probes in a 30-minute reaction. The end of the amplificate that is labeled with 5′dioxigenin now acts as the antigen for a specific antibody which is, in turn, coupled to the enzyme peroxidase. After addition of tetramethylbenzidine, a blue dye forms. Formation of the dye is stopped with 0.5 M sulfuric acid. At the same time, the color turns yellow because of the pH change. The intensity of the absorption is measured at 450 nm in an ELISA reader. 
     The following reagents are used to perform the ELISA: 
                                                                                                                                                                                 Hybridization buffer (2.5 × SSC)            2.5 × SSC   62.5 ml of 20 × SSC           (see below)       2 × Denhardts   20 ml of 50 × Denhardts           (see below)       10 mM Tris (Gibco, No. 15504-038)   5 ml of 1M Tris       1 mM EDTA (Fluka, No. 03699)   1 ml of 0.5M EDTA            Make up to 0.5 liter with double-distilled water and adjust to pH 7.5.       Wash buffer 1            1 × SSC   50 ml of 20 × SSC           (see below)       2 × Denhardts   40 ml of 50 × Denhardts           (see below)       10 mM Tris (Gibco, No. 15504-038)   10 ml of 1M Tris       1 mM EDTA (Fluka, No. 03699)   2 ml of 0.5M EDTA            Make up to 1 liter with double-distilled water and adjust to pH 7.5.       Wash buffer 2            100 mM Tris (Gibco, No. 15504-038)   12.15 g       150 mM NaCl (Merck, No. 6404.5000)   8.78 g       0.05% Tween 20 (Serva, No. 37470)   0.5 g       0.5% blocking reagent (Boehringer)   Dissolve 5 g in D1           (see below)           at 60° C.       10 μg/ml herring sperm   10 ml of the 10 mg/ml stock           solution            Dilute to 1 liter with double-distilled water and adjust to pH 7.5       Denaturation buffer            125 mM NaOH (Fluka, No. 71690)   0.5 g       20 mM EDTA (Fluka, No. 03699)   0.745 g            Make up to 0.1 liter with double-distilled water.       Coupling buffer            10 mM Tris (Gibco, No. 15504-038)   10 ml of 1M Tris       1 mM EDTA (Fluka, No. 03699)   2 ml of 0.5M EDTA       100 mM NaCl (Merck, No. 6404.5000)   5.88 g       0.15% Triton X 100 (Chemical storeroom)   15 ml            Make up to 1 liter with double-distilled water and adjust to pH 7.5.       Stop reagent (0.5M H 2 SO 4 )            95% H 2 SO 4     14 ml            Make up to 0.5 liter with double-distilled water.       50 × Denhardts            Ficoll 400 (Pharmacia Biotech,   5 g       No. 17-0400-01)       Polyvinylpyrrolidone (Sigma, No. P-2307)   5 g       Bovine serum albumin   5 g            Make up to 0.5 liter with double-distilled water.       20 × SSC            NaCl (Merck, No. 106404.1000)   350.36 g       Sodium citrate (trisodium citrate,   176.29 g       dihydrate, Fluka No. 71404)            Make up to 2 liters with double-distilled water and adjust to pH 7.0.       D 1            100 mM maleic acid (Fluka, No. 63190)   11.62 g       150 mM NaCl (Merck, No. 106404.1000)   8.76 g       NaOH (Fluka, No. 71690)   ca. 7.5 g            Make up to 2 liters with double-distilled water and adjust to pH 7.0.                    
ELISA Procedure:
 
     200 μl binding buffer and 1 μl probe are applied for each well. The microtiter plate is covered with an adhesive film and left to stand for two hours at room temperature. The PCR amplificates to be examined are thawed at room temperature, mixed with the denaturation buffer in the ratio of 1:1, and incubated for 10 minutes at room temperature. Then 10 ml of this probe is placed into the wells, which have been emptied in the meantime. In addition, 100 μl hybridization buffer is added to each well and incubated for 30 minutes at 37–60° C. To wash, the wells are emptied, filled with 200 ml wash buffer 1 which has been preheated to 37–60° C., and incubated for 2 minutes at the same temperature. This washing step is done three times. 
     After the wash buffer has been carefully removed, the Anti-Dig-POD-antibody (DAKO) is diluted 1:3000 (1 ml in 3 ml wash buffer 2), and 100 ml of this solution is placed into each of the dry wells. This arrangement is incubated in the incubator at 37° C. for 30 minutes. 
     Then the microtiter plate is washed three times with 200 ml wash buffer 2 per depression. Then 100 ml of the BM Blue dye (Boehringer) is added per well. After 15 minutes the reaction is stopped by addition of 100 ml 0.5 M H 2 SO 4 . The absorbance of the samples is measured in the ELISA reader. 
     The probes listed in Table 4 can be used to detect the species listed in the procedure described above. 
     EXAMPLE 5 
     General Usefulness of the DNA Regions Specified in this Patent for Detecting Bacteria 
     The ribosomal DNA regions specified here are suitable for detecting eubacteria, especially if they are combined with the 23 S–5 S ribosomal spacers. One skilled in the art can rapidly identify bacterial taxonomic units of his choice using the sequences under SEQ ID 1–530 or by focusing on the specified ribosomal DNA region. In the following, one possible way is exemplified which shows the general usefulness of this invention for all eubacterial species. 
     The path described here comprises essentially 3 steps. In the first step, a ribosomal region comprising approximately the last 330–430 nucleotides of the 23 S gene, the following transcribed spacer, and the ribosomal 5 S gene is amplified. As this region is of variable length in the various eubacterial species, it has a total length of 400 to about 750 nucleotides. If the DNA sequence is not yet known, it can be advantageous to determine it for the species to be detected and for some closely related species from which it must be distinguished. From a sequence comparison, one skilled in the art can easily determine the best oligonucleotides for the desired detection, e.g., serving as a PCR primer or as a probe. In this example, both primers and probes are selected in that manner. Alternatively, the sequences specified here can also be used directly for a wide spectrum of bacteria, especially if the stringency conditions for the PCR and/or for the hybridization are properly selected. 
     A) Amplification of Ribosomal DNA 
     The DNA segment to be used can be amplified from genomic bacterial DNA of the proteobacteria and many other bacterial classes with the primers SEQ ID 211 and 212. If other classes present problems in the DNA amplification, use of primers derived from DNA regions corresponding to SEQ ID 211 and 212 will be successful. 
     Genomic DNA is isolated from pure cultures of the bacteria listed in Table 5 by standard procedures which are themselves known. Quantities of about 1 to 100 ng each from these preparations are used in a PCR. The reaction solution has the following composition: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 genomic DNA 
                 1 
                 μl 
               
               
                   
                 H 2 O 
                 19.8 
                 μl 
               
               
                   
                 Buffer (10×)* 1   
                 2.5 
                 μl 
               
               
                   
                 dNTP (10 mM)* 2   
                 0.25 
                 μl 
               
               
                   
                 forward primer A (10 μM)* 3   
                 0.20 
                 μl 
               
               
                   
                 reverse primer (10 μM)* 3   
                 0.20 
                 μl 
               
               
                   
                 MgCl 2   
                 0.75 
                 μl 
               
               
                   
                 Taq polymerase (5 U/μl)* 1   
                 0.3 
                 μl 
               
               
                   
                   
               
               
                   
                 * 1 Buffer and enzyme from Biomaster or any other source. 
               
               
                   
                 * 2 Nucleotides from Boehringer Mannheim or any other source. 
               
               
                   
                 * 3 Equimolar quantities of primers. 
               
               
                   
                 In the case of mixtures, each forward and reverse primer has a total final concentration of 10 μM. 
               
             
          
         
       
     
     The PCR is done in a Perkin Elmer 9600 Thermocycler with the thermoprofile shown below: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 initial denaturation 
                 95° C. 
                  5 minutes 
               
               
                   
                 amplification (35 cycles) 
                 92° C. 
                  1 minute 
               
               
                   
                   
                 52° C. 
                  1 minute 
               
               
                   
                   
                 72° C. 
                 30 seconds 
               
               
                   
                 final synthesis 
                 72° C. 
                  5 minutes 
               
               
                   
                   
               
             
          
         
       
     
     Examples of genomic DNA which can be used for amplification are listed in Table 5. 
     B) Genus-Specific and Species-Specific Amplification of a Subregion of the Product from A. 
     The DNA product amplified in A) can be used directly to detect bacteria, especially if specific probes are used. It can be advantageous to amplify primarily a subregion of this sequence if the process is intended to provide limitation to a smaller systematic unit of the bacteria, such as species, genera or families. At least part of the differentiating ability can then be provided already by the amplification primer. The region amplified in A) provides many subregions with specific differentiation capabilities. One skilled in the art can easily recognize those regions by comparing the sequences of bacteria to be identified with closely related bacteria. 
     In this example, the beginning of the 23 S–5 S transcribed spacer and the end of it were selected as regions for specific primers. The actual sequences and the origin of the primer are summarized in Table 5. Comparison of the sequences shows that they basically provide a species-specific detection already. The primers for the  Vibrio  species are exceptions, allowing a genus-specific detection. In the forward primers, the sequence CGAAG . . . TTTT is conserved, in particular for enterobacteria, and in the reverse primers the sequence AACAGMTTT is conserved. Now there are two possibilities for expanding the specificity of the primers to genera and groups of genera, of the Enterobacteria, for instance. One is to lower the annealing temperatures in the PCR. The other is to shift the sequences for the forward primers toward the 23 S gene, and those for the reverse primers toward the 5 S gene. The result is primers in which the sequences are less variable by species. The actual design, then, can be directed to the requirements for detection. Here, we provide examples of the species-specific detection with the primers of Table 5 by PCR amplification. 
     Genomic DNA is isolated from pure cultures of the bacteria listed in Table 5 by standard procedures which are themselves known. Quantities of about 1 to 100 ng each from these preparations are used in a PCR. The reaction solution has the following composition: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 genomic DNA 
                 1 
                 μl 
               
               
                   
                 H 2 O 
                 19.8 
                 μl 
               
               
                   
                 Buffer (10×)* 1   
                 2.5 
                 μl 
               
               
                   
                 dNTP (10 mM)* 2   
                 0.25 
                 μl 
               
               
                   
                 forward primer (10 μM)* 3   
                 0.20 
                 μl 
               
               
                   
                 reverse primer* (10 μM)* 3   
                 0.20 
                 μl 
               
               
                   
                 MgCl 2   
                 0.75 
                 μl 
               
               
                   
                 Taq polymerase (5 U/μl)* 1   
                 0.3 
                 μl 
               
               
                   
                   
               
               
                   
                 * 1 Buffer and enzyme from Biomaster or any other source. 
               
               
                   
                 * 2 Nucleotides from Boehringer Mannheim or any other source. 
               
               
                   
                 * 3 Forward primer A and reverse primers* are listed in Table 5. In the case of mixtures, each forward and reverse primer has a total final concentration of 10 μM. Reverse primers* have the sequence complementary to the reverse primers shown in Table 5. 
               
             
          
         
       
     
     The PCR is done in a Perkin Elmer 9600 Thermocycler with the thermoprofile shown below: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 initial denaturation 
                 95° C. 
                  5 minutes 
               
               
                   
                 amplification (35 cycles) 
                 92° C. 
                  1 minute 
               
               
                   
                   
                 *45–72° C. 
                  1 minute 
               
               
                   
                   
                 72° C. 
                 30 seconds 
               
               
                   
                 final synthesis 
                 72° C. 
                  5 minutes 
               
               
                   
                   
               
               
                   
                 *The annealing temperature can be determined according to the generally used formulas for PCR primers. 
               
             
          
         
       
     
     Table 5 shows the result of the amplification, i.e. the species-specific detection of bacteria using the primers of Table 5 leads to identification of the bacteria assigned to those primers in this table. On the other hand, use of more general primers, the design of which was described before, can lead to detection of all enterobacterial genera or to detection of all the genera from the γ branch of the proteobacteria. 
     C) Making the Detection More Specific by Using Primers or Probes from the 23 S–5 S Ribosomal Spacer. 
     If DNA of higher taxonomic units was amplified in steps A) and/or B), then further differentiation of the detection can be accomplished by selection of probes. A more variable DNA region, such as a central region of the 23 S–5 S transcribed spacer can be used for species-specific detection. The probes can be integrated into a chip or used in the lightcycler technology or in an ELISA. In the latter case, the ELISA protocol in Example 4 can be used. Then the results of the species-specific detection of bacteria correspond to the selection of the 23 S–5 S transcribed spacer, because it has mostly a species-specific sequence region. When the primers from Table 5 are used, with use of the corresponding spacer (column SEQ ID from Table 5), then the species listed in that table can be identified. 
     Explanations of Concepts Used: 
     Derivation of DNA Sequences 
     A polynucleotide or oligonucleotide to be used for detection of taxonomic units can be found and developed by deriving it from one or more DNA sequences. In the case of multiple DNA sequences, alignment of the sequences, i.e., a comparison, is advantageous. Derived oligonucleotides may be identical to the original sequence. They may also be a consensus of numerous variables. In that case, the nucleotides of the polymer are selected according to the components most frequently used, or prevalent, at a certain position of the sequences analyzed. It is also possible to select variables in a sequence being developed according to the definition given for “nucleotide”. The DNA or RNA polymers resulting from these variable sequences are, then, a mixture of molecules exhibiting all the nucleotides allowed at the positions of the variables. 
     Analogous DNA Sequences: 
     Analogous DNA sequences have the same function, or a similar location, as a specified sequence, but cannot be traced back to the same phylogenetic origin. One example is the transcribed spacer between 5 S rDNA and 23 SD rDNA, if it exhibits no similarity with a transcribed spacer at the same location which is being compared with it. That is possible because it is often so variable in distantly related organisms that it is no longer possible to establish its phylogenetic evolution or homology. The transcribed spacer above, though, is clearly definable as a DNA sequence and in its function as a transcribed spacer, or in its location, because it begins at the end of the coding region of the 23 S rDNA and ends at the beginning of the 5 S rDNA. 
     Adjacent Genes: 
     Genes are adjacent if they are not separated by any other gene or if that is the case for two particular genes for most of the species studied. Separation is said to exist only if there is another gene between two other genes. 
     Enterobacteria 
     The Enterobacteria are a family of the γ-branch of the proteobacteria. The concept involves all the taxonomic units of the family, especially the genera  Alterococcus, Aquamonas, Aranicola, Arsenophonus, Brenneria, Budvicia, Cedecea, Calymmatobacterium, Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella, Hafnia, Klebsiella, Kluyvera, Koserella, Leclercia, Moellerella, Morganella, Pantoea, Phlomobacter, Photorhabdus, Plesiomonas, Proteus, Providencia, Rahnella, Salmonella, Serratia, Shigella, Wigglesworthia, Xenorhabdus, Yersinia , and  Yokenella.    
     Eubacteria: 
     The Eubacteria, along with the Archaebacteria, make up a kingdom of the Prokaryotes. Here “bacteria” and “eubacteria” are used synonymously. The concept includes all the taxonomic units within this kingdom. The Eubacteria include, for instance, the Aquificales, Aquificaceae,  Desulfurobacterium  group, Chlamydiales, Verrumicrobia group, Chlamydiaceae, Simkaniaceae, Waddliaceae, Verrumicrobia, Verrumicrobiales,  Coprothermobacter  group, Cyanobacteria, Chroococcales, Nostocales, Oscillatoriales, Pleurocapsales, Prochlorophytes, Stigonematales, Cytophagales, the green sulfur bacteria group, Bacteroidaceae, Cytophagaceae, Flavobacteriaceae,  Flexibacter  group,  Hymenobacter  group,  Rhodothermus  group,  Saprospira  group, Sphingobacteriaceae, Succinovibrionaceae, green sulfur bacteria,  Fibrobacter, Acidobacterium  group,  Fibrobacter  group, Firmicutes, Actinobacteria, Acidomicrobidae, Actinobacteridae, Coriobacteridae, Rubrobacteridae, Sphaerobacteridae,  Bacillus  group,  Clostridium  group,  Lactobacillus  group,  Streptococcus  group, Clostridiaceae, Haloanaerobiales,  Heliobacterium  group, Mollicutes,  Sporomusa  branch,  Syntrophomonas  group,  Thermoanaerobacter  group,  Flexistipes  group, Fusobacteria, green non-sulfur bacteria, Chloroflexaceae group, Chloroflexaceae, photosynthetic  Flexibacteria, Holophaga  group,  Nitrospira  group, Planctomycetales, Planctomycetaceae, Proteobacteria, purple non-sulfur bacteria, alpha subdivision of the proteobacteria, beta subdivision of the proteobacteria, gamma subdivision of the proteobacteria, delta/epsilon subdivision of the proteobacteria, Spirochetales, Leptospiraceae, Spirochaetaceae,  Synergistes  group,  Thermodesulfobacterium  group, Thermotogales,  Thermus  group or the  Deinococcus  group. 
     Gene: 
     The gene comprises the open reading frame or coding region of a DNA. Thus it codes solely for a single protein. The cistron is also a gene, but it, along with other cistrons, is on a mRNA. DNA regions which regulate transcription of the gene, such as promoters, terminators, and enhancers, are also part of the gene. When, in this patent, we speak, in a simplifying manner of the 23 S rDNA gene and the 5 S rDNA gene, this is based on the usual designations. According to our definition, though, the 23 S rDNA gene or the 5 S rDNA gene is not a gene but an independent functional DNA segment, because it does not code for a protein and cannot be subdivided into codons. 
     Transcribed Spacer: 
     The transcribed spacer, on which we focus here, lies behind the coding region of the 23 S rDNA gene and before the coding region of the 5 S rDNA gene. In its systematic classification, it has a special position. Because it is transcribed, and thus is part of the mRNA and a biologically inactive precursor molecule, preRNA, it is not part of the intergene region. The precursor molecule is converted into a biologically active molecule in the ribosomal context by excising the transcribed spacer. On the other hand, it cannot be assigned functionally or phylogenetically to the 23 S gene or the 5 S gene. As the gene concept apparently cannot be utilized for classification in this case, let the “transcribed spacer” of the ribosomal operon be considered an independent functional DNA (RNA) class equivalent to the “gene” and the “intergenic region”. 
     Homologous DNA Sequences 
     DNA or RNA sequences are homologous if they have the same phylogenetic origin. That may be recognizable by the fact that at least 40% of the nucleotides in a DNA segment are identical. There may be variable pieces in a large DNA segment. In that case it is sufficient for the phylogenetic relation to be shown by presence of a sequence 25 nucleotides long, which is at least 60% identical with another sequence, 25 nucleotides long, of the DNA being compared. Also, homologous sequences can frequently best be recognized by comparison with closely related organisms. To recognize homology of sequences of more distantly related organisms, it is then necessary to do a step-by-step comparison with sequences of species which bridge the separation to the distantly related phylogenetic species. 
     Identical DNA Sequences/Percent Identity 
     Subsequences of a larger polynucleotide are considered to determine the identity (in the sense of complete agreement, equivalent to 100% identity) of DNA or RNA sequences. These subsequences comprise 10 nucleotides, and are identical if all 10 components are identical in two comparison sequences. The nucleotides thymidine and uridine are considered identical. All the possible fragments of a larger polynucleotide can be considered as subsequences. 
     The identity is 90% if 9 of 10 nucleotides, or 18 or 20 nucleotides, are the same in a section on the two sequences being compared. 
     As an example, consider two polynucleotides made up of 20 nucleotides, which differ at the 5 th  component. In a sequence comparison, then one would find six 10-element nucleotides which are identical and 5 which are not identical because they differ in one component. 
     The identity can also be determined by degrees, with the unit reported being a percentage. To determine the degree of identity such subsequences are considered that comprise at least the length of the sequence actually used, e.g. as a primer, or 20 nucleotides. 
     As an example, we compare polynucleotide A with a length of 100 nucleotides and polynucleotide B with a length of 200 nucleotides. A primer is derived from polynucleotide B with a length of 14 nucleotides. To determine the degree of identity, polynucleotide A is compared with the primer over its entire length. If the sequence of the primer occurs in polynucleotide A, but with a difference in one component, then we have a fragment with a degree of identity of 13/14, or 92.3%. 
     As a second example, the two polynucleotides above, A and B, are compared in their entirety. In this case, all the possible comparison windows with lengths of 20 nucleotides are applied and their degrees of identity are determined. Then if nucleotides numbered 50–69 of polynucleotides A and B are identical except for nucleotide number 55, then these fragments have a degree of identity of 19/20 or 95%. 
     Conserved and Variable Primers 
     Conserved primers are nucleotides which hybridize with conserved DNA or RNA regions. The concept ‘conserved’ characterizes the evolutionary variability of a nucleotide sequence for species of various taxonomic units. Therefore it is a measure of comparison. Depending on which sequence is used for comparison, a region or primer can be conserved or variable. Characterization of a primer as “conserved” or “variable” is accomplished by means of directly adjacent or overlapping regions with respect to the of hybridization target, which have the same length as the primer. Therefore one can select comparison sequences from the same organism, or homologous or similar segments from different organisms. When two sequences are compared, one is conserved if it is at least 95% identical with the comparison sequence, or variable if it is less than 95% identical. 
     Nested Primers 
     Nested primers are used particularly in consensus PCR. These are primers which amplify a fragment of an already amplified polynucleotide. Therefore nested primers hybridize with a region within an already multiplied DNA or RNA target molecule. Amplification with nested primers can be done as frequently as desired, giving successively smaller amplification products. 
     Hybridization of DNA or RNA 
     Two identical or similar nucleotide fragments can hybridize with each other to form a double strand. Such hybridization does not occur only between DNA, RNA, or PNA single strands. It is also possible for hybrid molecules to form between DNA and RNA, DNA and PNA, RNA and PNA, etc. There are numerous factors which determine whether two polynucleotides hybridize. Hybridization can take place in a temperature range of, preferably, 37–60° C. Hybridization can also occur in discrete hybridization and washing steps. Example 4) presents experimental parameters to make hybridization conditions more specific. Specific hybridization takes place if only a single hybridization with the desired target sequence occurs with the probe used and not with any other DNA which is also in the sample. 
     Combinations in Use of Nucleotides 
     Primers, probes, DNA fragments, subregions of polynucleotides or oligonucleotides can be used in many combinations. Possibilities include, for instance, arbitrary combination of two primers from a group of primers; arbitrary selection of one probe from a group of sequences; and selection of primers from the same group of sequences. In the latter cases the primer and probe(s) may be identical or different. Primers or probes can also be made up of two or more DNA fragments, with all possible variations in the composition being eligible. Combinations are also possible in the sequence of distinct PCR steps with different primers and the use of probes. 
     Consensus PCR 
     A consensus PCR is carried out with consensus primers. These are able to amplify the DNA of at least 2 taxonomic units (of all taxonomic units in the ideal case). In subsequent analysis steps, the identity of the amplified DNA is determined. For this purpose, either other PCR steps are done, which discriminate between smaller taxonomic units with variable nested primers if necessary, or the final determination of a laxonomic unit can be done with specific probes rather than with variable primers. 
     Nucleotides 
     Nucleotides are the building blocks of DNA or RNA. The abbreviations mean: G=guanosine, A=adenosine, T=thymidine, C=cytidine, R=G or A; Y=C or T; K=G or T; W=A or T; S=C or G; M=A or C; B=C, G or T; D=A, G or T; H=A, C or T; V=A, C, or G; N=A, C, G. or T; I=inosine. 
     Taxonomic Units 
     Taxonomic units of bacteria are all the known taxonomic subdivisions, such as kingdoms, classes, phyla, orders, families, genera, species, strains, intermediates of those taxonomic units such as subclasses, suborders, subfamilies, etc.; or groups of these taxonomic units. 
     DETAILED DESCRIPTION OF THE INVENTION 
     This invention comprises essentially 5 partial aspects which reflect the invention in its general form and in its special aspects:
         strategic selection of DNA target regions using adjacent genes   description of use of a ribosomal DNA region from the end of the 23 S rDNA, the transcribed spacer, and parts of the 5 S rDNA to detect all bacteria   provision of primers and probes for many bacteria   detection of the families of the enterobacteria and their members   use of a consensus PCR to detect all bacteria
 
Strategic Selection of DNA Target Regions Using Adjacent Genes
       

     The invention consists in the use of portions of adjacent genes to detect taxonomic units, i.e., kingdoms, classes, phyla, families, genera and strains, as well as intermediate forms of these units. The advantage of the invention is that DNA regions which span two genes are very heterogeneous with respect to variability. That has been found, for instance, with the ribosomal operons, especially the 23 S/5 S rDNA segment. Because of the presence of very strongly conserved regions and very poorly conserved regions, one skilled in the art is enabled to detect all possible closely and even distantly related organisms. 
     Description of Use of a Ribosomal DNA Region from the End of the 23 S rDNA, From the Transcribed Spacer, and From Parts of the 5 S rDNA to Detect all Bacteria 
     In particular, a 23 S–5 S rDNA region comprising about 400–750 nucleotides can be used to detect bacteria. The latter region consists of about 330–430 nucleotides of the terminal region of the 23 S rDNA, the adjoining transcribed spacer, and the 5 S rDNA gene. In individual cases, a t-RNA gene can also be inserted into the spacer and used for the detection. The region described corresponds to the nucleotides 2571–3112 of the SEQ ID 1, which represents the 23 S and 5 S rDNA genes of  Escherichia coli . The homologous regions, and those corresponding to the above region, from other bacteria can be determined by a sequence comparison known to those skilled in the art. The beginning of the above-described region at the terminus of the 23 S rDNA gene and the end of the 5 S rDNA genes can be determined easily by comparing the ribosomal DNA sequences of two species A and B, especially for members of the same families, or even orders or phyla. Should this not be as easy for a comparison of species A and a more distantly related species C, one arrives at the desired result by making a comparison between the sequences of species B and C, in which B and C should be closely related to each other. In this way, by a series of separate sequence comparisons, it is possible to determine the homogeneous ribosomal regions of the 23 S rDNA, the transcribed spacer, and the 5 S rDNA of all Eubacteria. Because of the variability of individual subregions, length differences of several hundred nucleotides can occur. In addition, this invention allows use of subregions of the region described above. Table 6 describes a large portion of these regions. 
     Provision of/Providing Primers and Probes for Many Bacteria 
     Along with the general description of the useful rDNA region, sequences (SEQ ID 1–530) are also provided, which can be used to detect bacteria. Depending on the particular objective, the polynucleotides occurring in SEQ ID 1–530 can be used completely, or fragments of the sequence can be used. The sequences specified in SEQ ID 1–530 are derived from the previously described region of the 23 S rDNA gene, transcribed spacer, and 5 S rDNA gene. 
     In the technical execution, organisms can be detected by means of the DNA regions and sequences specified for that purpose, using probes and/or primers. Primers are nucleotides which act as starter molecules for the amplification. They deposit on the target sequence, so that the region is synthesized anew using a polymerase. Their specificity can be adjusted by the degree of identity of the primer with the target sequence. The taxonomic specificity is also determined by the selection of the target sequence within the ribosomal region described here (see also Table 6). Primers can thus be used in different ways: For instance, it is possible to amplify the entire region corresponding to  FIG. 2 , or homologous to the nucleotides number 2571–3112 of the SEQ ID 1 ( E. coli ) with primers SEQ ID 211 and 212. A mixture of more than two primers can also be used to optimize the amplification. Moreover it is possible to select the primer so that only the DNA of certain bacteria is amplified. In this case, then, there are two kinds of information in the case of positive amplification: First, they show the presence of the bacteria sought; and second, they show the identity of the bacteria. By means of sequential amplification steps with nested primers, the information obtained at the end of the DNA synthesis can be adjusted according to the requirements. 
     In a distinct step, the DNA, which ideally has previously been amplified, is bound to probes, concentrated, and detected. Probes are oligonucleotides or polynucleotides which can bind to single-stranded DNA segments. The affinity of the probes to the target sequence is determined by their degree of identity with it. The hybridization conditions also have a significant effect. That is, the buffer salt concentration, the incubation time, and the incubation temperature must be optimized. One skilled in the art can rapidly optimize those parameters using current methods. Exemplary hybridization conditions are given in the examples. Probes, just like primers, can work in two ways. First, they can show the presence of bacterial DNA or amplification products. Second, they can contribute to the detection of the DNA of specific bacteria. In this duality of their function they resemble the primers. Accordingly, the task of identification of organisms can be divided between primers and probes. Also, the probes, like the primers, derive from freely selectable regions of the terminal region of the 23 S rDNA, of the transcribed spacer, of the 5 S rDNA, or from the entire region. 
     One special advantage of this invention is that the ribosomal region selected according to  FIG. 2  is be composed heterogeneously of very variable and very conserved regions, over an extremely broad range. As there are very many combinations in utilization of subregions, e.g., as shown in Table 6, this invention offers the potential of detecting all bacterial species and taxonomic units. 
     Detection of the Family of the Enterobacteria and their Members 
     Bacterial families such as the Enterobacteriaceae can be detected by using the DNA target regions characterized in this document (Example 1). The enterobacteria are a homogeneous taxonomic unit of the γ branch of the proteobacteria or purple bacteria. They are of particular interest because they include many pathogenic bacteria, such as  Escherichia coli  (EHEC, etc.),  Shigella, Salmonella , and  Yersinia . Thus they are suitable marker organisms for examining the hygienic status of foods. In clinical microbiology, detection of enterobacteria can be an initial step in narrowing down or identifying pathogenic microorganisms. From the list contained in this work, for instance, the primer SEQ ID 2–25, in various combinations, is usable for identifying the enterobacteria as the family. Many of the sequences listed are also suitable for identifying individual members of the enterobacteria, i.e., genera, species and strains. Other sequences are also produced for the other taxonomic units of the proteobacteria, especially the entire γ branch, as well as for the Firmicutes. Description of the ribosomal region as shown in  FIG. 2  shows another way in which one skilled in the art can easily obtain more sequences so as to detect all the Eubacteria. 
     Use of a Consensus PCR to Detect all Bacteria 
     One special advantage of our invention is that the DNA target region, as described in  FIG. 2 , can be detected in an ideal manner in a consensus PCR. One significant prerequisite for the experimental applicability of this method is that the sequences become increasingly variable within a target region to be amplified. The region of the ribosomal operon which we have characterized has such a configuration for all the species investigated. 
     The plan for the consensus PCR is outlined in  FIG. 8 . As a general rule, a “master fragment” is amplified first. That can be the same as the complete fragment as shown in  FIG. 2 , or a part of it. Now if there are various microorganisms to be identified in a sample, this fragment is amplified for all of them. Finally, the individual organisms are identified with specific probes and/or in combination with more PCR steps. The detection with probes can even be miniaturized and accomplished on chips. Alternatively, detection can be done in the classical ELISA procedure. The components for bacterial detection can be prepared in the form of a kit. 
     Fluorescent dyes are particularly advantageous for detection. They can be coupled to the primers or to the probes. However, non-fluorescent dyes are also used often, particularly in the ELISA or the Southern Blot procedures. Genetrack and Light Cycler technology provides another possibility for detection. In principle, all these methods offer the option of quantitative determination. Thus by evaluating the detection signal it is also possible to ultimately draw conclusions about the number of bacteria in a sample. 
     Detection of bacteria with this invention can be done in an experimental context that is well known to one skilled in the art. For instance, bacteria can first be enriched in a suitable medium before detection. In working with foods, physical separation steps such as centrifugation or sedimentation are advantageous. It is also possible to enrich the bacteria in such a way that it is later possible to draw conclusions about their initial number. Furthermore, one can do threshold value tests with respect to the bacterial count. All in all, then, quantitative or semiquantitative determination of microorganisms is possible. 
     The (enriched) bacteria are broken up to isolate the genomic DNA. The procedures for cell disintegration that are well known to one skilled in the art are often based on physical (glass beads, heat) and chemical (NaOH) influences. It is also possible, though, to use cells directly in a PCR to detect DNA. Moreover it can also be advantageous to purify the genomic DNA, especially if it is distributed through a food matrix. These procedures are also known to those skilled in the art. DNA purification kits are also commercially available. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Detection of  enterobacteria  excluding other bacteria (Example 1) 
               
             
          
           
               
                 No. 
                 Species 
                 Strain 
                 Detection 
               
               
                   
               
             
          
           
               
                 1 
                 
                   Budvicia aquatilis 
                 
                 DSM 5025 
                 + 
               
               
                 2 
                 
                   Buttiauxella agrestis 
                 
                 DSM 4586 
                 + 
               
               
                 3 
                 
                   Cedecea davisae 
                 
                 DSM 4568 
                 + 
               
               
                 4 
                 
                   Citrobacter koser 
                 
                 DSM 4595 
                 + 
               
               
                 5 
                 
                   Erwinia carotovora 
                 
                 DSM 30168 
                 + 
               
               
                 6 
                 
                   Erwinia chrysanthemi 
                 
                 DSM 4610 
                 + 
               
               
                 7 
                 
                   Ewingella americana 
                 
                 DSM 4580 
                 + 
               
               
                 8 
                 
                   Enterobacter agglomerans 
                 
                 B-5081-i 
                 + 
               
               
                 9 
                 
                   Enterobacter aerogenes 
                 
                 DSM 30053 
                 + 
               
               
                 10 
                 
                   Enterobacter sakazakii 
                 
                 DSM 4485 
                 + 
               
               
                 11 
                 
                   Enterobacter intermedius 
                 
                 DSM 4581 
                 + 
               
               
                 12 
                 
                   Enterobacter cloacae 
                 
                 DSM 30054 
                 + 
               
               
                 13 
                 
                   E. coli 
                 
                 BC 7883 
                 + 
               
               
                 14 
                 
                   E. coli 
                 
                 H123 
                 + 
               
               
                 15 
                 
                   E. coli 
                 
                 BC 7884 
                 + 
               
               
                 16 
                 
                   E. coli 
                 
                 BC 7885 
                 + 
               
               
                 17 
                 
                   E. hermanii 
                 
                 B-4943a 
                 + 
               
               
                 18 
                 
                   E. coli 
                 
                 ATCC 8739 
                 + 
               
               
                 19 
                 
                   Hafnia alvei 
                 
                 DSM 30163 
                 + 
               
               
                 20 
                 
                   Klebsiella pneumoniae 
                 
                 ATCC 13883 
                 + 
               
               
                 21 
                 
                   Klebsiella pneumoniae 
                 
                 DSM 2026 
                 + 
               
               
                 22 
                 
                   Klebsiella planticola 
                 
                 DSM 4617 
                 + 
               
               
                 23 
                 
                   Klebsiella oxytoca 
                 
                 DSM 5175 
                 + 
               
               
                 24 
                 
                   Kluyvera cryocrescens 
                 
                 DSM 4583 
                 + 
               
               
                 25 
                 
                   Morganella morganii 
                 
                 DSM 30164 
                 + 
               
               
                 26 
                 
                   Plesiomonas shigelloides 
                 
                 DSM 8224 
                 + 
               
               
                 27 
                   Pantoea  ssp. 
                 B-5200 
                 + 
               
               
                 28 
                 
                   Pantoea dispersa 
                 
                 DSM 30073 
                 + 
               
               
                 29 
                 
                   Proteus rettgeri 
                 
                 DSM 1131 
                 + 
               
               
                 30 
                 
                   Proteus rettgeri 
                 
                 ATCC 14505 
                 + 
               
               
                 31 
                 
                   Providencia stuartii 
                 
                 DSM 4539 
                 + 
               
               
                 32 
                 
                   Rahnella aquatilis 
                 
                 DSM 4594 
                 + 
               
               
                 33 
                 
                   Rahnella aquatilis 
                 
                 DSM 4594 
                 + 
               
               
                 34 
                 
                   Serratia proteamaculans 
                 
                 DSM 4487 
                 + 
               
               
                 35 
                 
                   Serratia ficaria 
                 
                 DSM 4509 
                 + 
               
               
                 36 
                 
                   Serratia plymutica 
                 
                 DSM 49 
                 + 
               
               
                 37 
                 
                   Serratia rubidea 
                 
                 DSM 4480 
                 + 
               
               
                 38 
                 
                   Serratia marcescens 
                 
                 DSM 1636 
                 + 
               
               
                 39 
                 
                   Salmonella bongori 
                 
                 DSM 7952 
                 + 
               
               
                 40 
                 
                   Yersinia pseudotuberculosis 
                 
                 DSM 8992 
                 + 
               
               
                 41 
                 
                   Yersinia pseudotuberculosis 
                 
                 DSM 8992 
                 + 
               
               
                 42 
                 
                   Yersinia enterolytica 
                 
                 DSM 4790 
                 + 
               
               
                 43 
                 
                   Acinetobacter calcoaceticus 
                 
                 DSM 590 
                 − 
               
               
                 44 
                 
                   Aeromonas hydrophila 
                 
                 DSM 6173 
                 − 
               
               
                 45 
                 
                   Aeromonas enteropelogenes 
                 
                 DSM 6394 
                 − 
               
               
                 46 
                   Fransilla tularensis  Isolat 
                 F16 
                 − 
               
               
                 47 
                 
                   Franzisella philomiragia 
                 
                 DSM 7535 
                 − 
               
               
                 48 
                 
                   Moraxella catarrhalis 
                 
                 DSM 9143 
                 − 
               
               
                 49 
                 
                   Pasteurella pneumotropica 
                 
                 B-2397 A 13 
                 − 
               
               
                 50 
                 
                   Pseudomonas beyjerinkii 
                 
                 DSM 7218 
                 − 
               
               
                 51 
                 
                   Vibrio fischeri 
                 
                 DSM 507 
                 − 
               
               
                 52 
                 
                   Vibrio alginolyticus 
                 
                 DSM 2171 
                 − 
               
               
                 53 
                 
                   Vibrio proteolyticus 
                 
                 DSM 30189 
                 − 
               
               
                 54 
                 
                   Vibrio paramaemolytiucs 
                 
                 DSM 10027 
                 − 
               
               
                 55 
                 
                   Vibrio harveyi 
                 
                 DSM 6104 
                 − 
               
               
                 56 
                 
                   Xanthomonas maltophila 
                 
                 BC 4273 
                 − 
               
               
                 57 
                 
                   Achromobacter xylosa 
                 
                 DSM 2402 
                 − 
               
               
                 58 
                   Alcaligenes  spp 
                 DSM 2625 
                 − 
               
               
                 59 
                 
                   Alcaligenes latus 
                 
                 DSM 1122 
                 − 
               
               
                 60 
                 
                   Brucella neotomae 
                 
                 ATCC 25840 
                 − 
               
               
                 61 
                 
                   Brucella ovis 
                 
                 ATCC 23459 
                 − 
               
               
                 62 
                 
                   Enterococcus casseliflavus 
                 
                 DSM 20680 
                 − 
               
               
                 63 
                   Flavobacterium  sp 
                 ATCC 27551 
                 − 
               
               
                 64 
                 
                   Flavobacterium resinovorum 
                 
                 DSM 7438 
                 − 
               
               
                 65 
                 
                   Flavobacterium johnsonii 
                 
                 DSM 2064 
                 − 
               
               
                 66 
                 
                   Flavobacterium flavense 
                 
                 DSM 1076 
                 − 
               
               
                 67 
                 
                   Lactobacillus bifermentans 
                 
                 BC 8463 
                 − 
               
               
                 68 
                 
                   Pseudomonas paucimobilis 
                 
                 DSM 1098 
                 − 
               
               
                 69 
                 
                   Pseudomonas cepacia 
                 
                 DSM 3134 
                 − 
               
               
                 70 
                 
                   Sphingobacterium multivorans 
                 
                 DSM 6175 
                 − 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Detection of  Pantoea dispersa  excluding other bacteria (Example 2) 
               
             
          
           
               
                 No. 
                 Species 
                 Detection 
               
               
                   
               
             
          
           
               
                 1 
                 
                   Pantoea dispersa 
                 
                 + 
               
               
                 2 
                 
                   Budvicia aquatica 
                 
                 − 
               
               
                 3 
                 
                   Buttiauxella agrestis 
                 
                 − 
               
               
                 4 
                 
                   Enterobacter agglomerans 
                 
                 − 
               
               
                 5 
                 
                   Erwinia carotovora 
                 
                 − 
               
               
                 6 
                 
                   Erwinia crysanthemi 
                 
                 − 
               
               
                 7 
                 
                   Escherichia coli 
                 
                 − 
               
               
                 8 
                 
                   Escherichia vulneris 
                 
                 − 
               
               
                 9 
                 
                   Escherichia hermannii 
                 
                 − 
               
               
                 10 
                 
                   Hafnia alvei 
                 
                 − 
               
               
                 11 
                 
                   Klebsiella oxytoca 
                 
                 − 
               
               
                 12 
                 
                   Kluyvera cryoescens 
                 
                 − 
               
               
                 13 
                 
                   Morganella morganii 
                 
                 − 
               
               
                 14 
                 
                   Proteus mirabilis 
                 
                 − 
               
               
                 15 
                 
                   Proteus rettgeri 
                 
                 − 
               
               
                 16 
                 
                   Proteus stuartii 
                 
                 − 
               
               
                 17 
                 
                   Providencia stuartii 
                 
                 − 
               
               
                 18 
                 
                   Rahnella aquatilis 
                 
                 − 
               
               
                 19 
                 
                   Serratia ficaria 
                 
                 − 
               
               
                 20 
                 
                   Serratia fonticola 
                 
                 − 
               
               
                 21 
                 
                   Serratia marcescens 
                 
                 − 
               
               
                 22 
                 
                   Serratia plymuthica 
                 
                 − 
               
               
                 23 
                 
                   Serratia proteamaculans 
                 
                 − 
               
               
                 24 
                 
                   Serratia rubidea 
                 
                 − 
               
               
                 25 
                 
                   Yersinia enterolytica 
                 
                 − 
               
               
                 26 
                 
                   Yersinia peudotuberculosis 
                 
                 − 
               
               
                 27 
                 
                   Acinetobacter calcoaceticus 
                 
                 − 
               
               
                 28 
                 
                   Aeromonas enteropelogenes 
                 
                 − 
               
               
                 29 
                 
                   Aeromonas hydrophila 
                 
                 − 
               
               
                 30 
                 
                   Cedecea davisae 
                 
                 − 
               
               
                 31 
                 
                   Haemophilus influenzae 
                 
                 − 
               
               
                 32 
                 
                   Moraxella catarrhalis 
                 
                 − 
               
               
                 33 
                 
                   Pasteurella pneumotropica 
                 
                 − 
               
               
                 34 
                 
                   Stenotrophomonas multophila 
                 
                 − 
               
               
                 35 
                 
                   Vibrio alginolyticus 
                 
                 − 
               
               
                 36 
                 
                   Vibrio fisheri 
                 
                 − 
               
               
                 37 
                 
                   Vibrio harveyi 
                 
                 − 
               
               
                 38 
                 
                   Vibrio parahaemolyticus 
                 
                 − 
               
               
                 39 
                   Alcaligenes  sp. 
                 − 
               
               
                 40 
                 
                   Bacillus subtilis 
                 
                 − 
               
               
                 41 
                 
                   Brucella abortus 
                 
                 − 
               
               
                 42 
                 
                   Brucella ovis 
                 
                 − 
               
               
                 43 
                 
                   Flavobacterium resinovorum 
                 
                 − 
               
               
                 44 
                 
                   Pseudomonas paucimobilis 
                 
                 − 
               
               
                 45 
                 
                   Pseudomonas cepacia 
                 
                 − 
               
               
                 46 
                 
                   Ralstonia pickettii 
                 
                 − 
               
               
                 47 
                 
                   Sphingobacterium multivorum 
                 
                 − 
               
               
                 48 
                 
                   Sphingomonas paucimobilis 
                 
                 − 
               
               
                 49 
                 
                   Streptococcus faecalis 
                 
                 − 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Detection of a group of genera with the probe 
               
               
                 GTTCCGAGATTGGTT 
               
             
          
           
               
                 No. 
                 Species 
                 Detection 
               
               
                   
               
             
          
           
               
                 1 
                 
                   Rahnella aquatilis 
                 
                 + 
               
               
                 2 
                 
                   Serratia ficaria 
                 
                 + 
               
               
                 3 
                 
                   Serratia fonticola 
                 
                 + 
               
               
                 4 
                 
                   Serratia marcescens 
                 
                 + 
               
               
                 5 
                 
                   Serratia plymuthica 
                 
                 + 
               
               
                 6 
                 
                   Serratia proteamaculans 
                 
                 + 
               
               
                 7 
                 
                   Serratia rubidea 
                 
                 + 
               
               
                 8 
                 
                   Yersinia enterolytica 
                 
                 + 
               
               
                 9 
                 
                   Yersinia peudotuberculosis 
                 
                 + 
               
               
                 10 
                 
                   Budvicia aquatica 
                 
                 − 
               
               
                 11 
                 
                   Buttiauxella agrestis 
                 
                 − 
               
               
                 12 
                 
                   Enterobacter agglomerans 
                 
                 − 
               
               
                 13 
                 
                   Erwinia carotovora 
                 
                 − 
               
               
                 14 
                 
                   Erwinia crysanthemi 
                 
                 − 
               
               
                 15 
                 
                   Escherichia coli 
                 
                 − 
               
               
                 16 
                 
                   Escherichia vulneris 
                 
                 − 
               
               
                 17 
                 
                   Escherichia hermannii 
                 
                 − 
               
               
                 18 
                 
                   Hafnia alvei 
                 
                 − 
               
               
                 19 
                 
                   Klebsiella oxytoca 
                 
                 − 
               
               
                 20 
                 
                   Kluyvera cryoescens 
                 
                 − 
               
               
                 21 
                 
                   Morganella morganii 
                 
                 − 
               
               
                 22 
                 
                   Pantoea dispersa 
                 
                 − 
               
               
                 23 
                 
                   Proteus mirabilis 
                 
                 − 
               
               
                 24 
                 
                   Proteus rettgeri 
                 
                 − 
               
               
                 25 
                 
                   Proteus stuartii 
                 
                 − 
               
               
                 26 
                 
                   Providencia stuartii 
                 
                 − 
               
               
                 27 
                 
                   Acinetobacter calcoaceticus 
                 
                 − 
               
               
                 28 
                 
                   Aeromonas enteropelogenes 
                 
                 − 
               
               
                 29 
                 
                   Aeromonas hydrophila 
                 
                 − 
               
               
                 30 
                 
                   Cedecea davisae 
                 
                 − 
               
               
                 31 
                 
                   Haemophilus influenzae 
                 
                 − 
               
               
                 32 
                 
                   Moraxella catarrhalis 
                 
                 − 
               
               
                 33 
                 
                   Pasteurella pneumotropica 
                 
                 − 
               
               
                 34 
                 
                   Stenotrophomonas multophila 
                 
                 − 
               
               
                 35 
                 
                   Vibrio alginolyticus 
                 
                 − 
               
               
                 36 
                 
                   Vibrio fisheri 
                 
                 − 
               
               
                 37 
                 
                   Vibrio harveyi 
                 
                 − 
               
               
                 38 
                 
                   Vibrio parahaemolyticus 
                 
                 − 
               
               
                 39 
                   Alcaligenes  sp. 
                 − 
               
               
                 40 
                 
                   Bacillus subtilis 
                 
                 − 
               
               
                 41 
                 
                   Brucella abortus 
                 
                 − 
               
               
                 42 
                 
                   Brucella ovis 
                 
                 − 
               
               
                 43 
                 
                   Flavobacterium resinovorum 
                 
                 − 
               
               
                 44 
                 
                   Pseudomonas paucimobilis 
                 
                 − 
               
               
                 45 
                 
                   Pseudomonas cepacia 
                 
                 − 
               
               
                 46 
                 
                   Ralstonia pickettii 
                 
                 − 
               
               
                 47 
                 
                   Sphingobacterium multivorum 
                 
                 − 
               
               
                 48 
                 
                   Sphingomonas paucimobilis 
                 
                 − 
               
               
                 49 
                 
                   Streptococcus faecalis 
                 
                 − 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Specific probes for the detection of bacterial genera and species 
               
             
          
           
               
                   
                   
                 Probe 
                   
               
               
                   
                 No. 
                 SEQ ID 
                 Detection of Genus/Species 
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 96 
                 
                   Budvicia aquatica 
                 
               
               
                   
                 2 
                 97 
                 
                   Buttiauxella agrestis 
                 
               
               
                   
                 3 
                 98 
                 
                   Enterobacter agglomerans 
                 
               
               
                   
                 4 
                 99 
                 
                   Erwinia carotovora 
                 
               
               
                   
                 5 
                 100 
                 
                   Erwinia chrysanthemi 
                 
               
               
                   
                 6 
                 101 
                 
                   Escherichia coli 
                 
               
               
                   
                 7 
                 102 
                 
                   Escherichia hermannii 
                 
               
               
                   
                 8 
                 103 
                 
                   Escherichia vulneris 
                 
               
               
                   
                 9 
                 104 
                 
                   Hafnia alvei 
                 
               
               
                   
                 10 
                 105 
                 
                   Klebsiella oxytoca 
                 
               
               
                   
                 11 
                 106 
                 
                   Kluyvera cryoescens 
                 
               
               
                   
                 12 
                 107 
                 
                   Morganella morganii 
                 
               
               
                   
                 13 
                 108, 109 
                 
                   Pantoea 
                 
               
               
                   
                 14 
                 110 
                 
                   Proteus mirabilis 
                 
               
               
                   
                 15 
                 111 
                 
                   Proteus rettgeri 
                 
               
               
                   
                 16 
                 112 
                 
                   Providencia stuartii 
                 
               
               
                   
                 17 
                 113 
                 
                   Rahnella aquatilis 
                 
               
               
                   
                 18 
                 114 
                 
                   Serratia ficaria 
                 
               
               
                   
                 19 
                 115 
                 
                   Serratia fonticola 
                 
               
               
                   
                 20 
                 116 
                 
                   Serratia marcescens 
                 
               
               
                   
                 21 
                 117 
                 
                   Serratia plymuthica 
                 
               
               
                   
                 22 
                 118 
                 
                   Serratia proteamaculans 
                 
               
               
                   
                 23 
                 119 
                 
                   Serratia rubidea 
                 
               
               
                   
                 24 
                 120 
                 
                   Yersinia enterolytica 
                 
               
               
                   
                 25 
                 121 
                 
                   Yersinia pseudotuberculosis 
                 
               
               
                   
                 26 
                 122 
                 
                   Acinetobacter calcoaceticus 
                 
               
               
                   
                 27 
                 123 
                 
                   Aeromonas enteropelogenes 
                 
               
               
                   
                 28 
                 124 
                 
                   Aeromonas hydrophila 
                 
               
               
                   
                 29 
                 125 
                 
                   Cedecea davisae 
                 
               
               
                   
                 30 
                 126 
                 
                   Haemophilus influenzae 
                 
               
               
                   
                 31 
                 127 
                 
                   Moraxella catharralis 
                 
               
               
                   
                 32 
                 128 
                 
                   Pasteurella pneumotropica 
                 
               
               
                   
                 33 
                 129 
                 
                   Stenotrophomonas multophila 
                 
               
               
                   
                 34 
                 130 
                 
                   Vibrio alginolyticus 
                 
               
               
                   
                 35 
                 131 
                 
                   Vibrio fisheri 
                 
               
               
                   
                 36 
                 132 
                 
                   Vibrio harveyi 
                 
               
               
                   
                 37 
                 133 
                 
                   Vibrio parahaemolyticus 
                 
               
               
                   
                 38 
                 134 
                 
                   Vibrio proteolyticus 
                 
               
               
                   
                 39 
                 432 
                 
                   Salmonella typhi 
                 
               
               
                   
                 40 
                 433 
                 
                   Buchnera aphidocola 
                 
               
               
                   
                 41 
                 434 
                 
                   Pseudomonas stutzeri 
                 
               
               
                   
                 42 
                 435 
                 
                   Thiobacillus ferrooxidans 
                 
               
               
                   
                 43 
                 436 
                 
                   Agrobacterium vitis 
                 
               
               
                   
                 44 
                 437 
                 
                   Adalia bipunctata 
                 
               
               
                   
                 45 
                 438 
                 
                   Amycocalatopsis orientalis 
                 
               
               
                   
                 46 
                 439 
                 
                   Brucella 
                 
               
               
                   
                 47 
                 440 
                 
                   Bradyrhyzobium japonicum 
                 
               
               
                   
                 48 
                 441 
                 
                   Pseudomonas paucimobilis 
                 
               
               
                   
                 49 
                 442 
                 
                   Rhodobacter sphaeroides 
                 
               
               
                   
                 50 
                 443 
                 
                   Rickettsia prowazekii 
                 
               
               
                   
                 51 
                 444 
                 
                   Pseudomonas cepacia 
                 
               
               
                   
                 52 
                 445 
                 
                   Ralstonia pickettii 
                 
               
               
                   
                 53 
                 446 
                 
                   Campylobacter jejuni 
                 
               
               
                   
                 54 
                 447 
                 
                   Helicobacter pylori 
                 
               
               
                   
                 55 
                 448 
                 
                   Actinoplanes utahensis 
                 
               
               
                   
                 56 
                 449 
                 
                   Bacillus halodurans 
                 
               
               
                   
                 57 
                 450 
                 
                   Bacillus subtilis 
                 
               
               
                   
                 58 
                 451 
                 
                   Clostridium tyrobutyricum 
                 
               
               
                   
                 59 
                 452 
                 
                   Frankia 
                 
               
               
                   
                 60 
                 453 
                 
                   Microbispora bispora 
                 
               
               
                   
                 61 
                 454 
                 
                   Mycobacterium leprae 
                 
               
               
                   
                 62 
                 455 
                 
                   Mycobacterium smegmatis 
                 
               
               
                   
                 63 
                 456 
                 
                   Mycobacterium tuberculosis 
                 
               
               
                   
                 64 
                 457 
                 
                   Mycoplasma gallisepticum 
                 
               
               
                   
                 65 
                 458 
                 
                   Propionibacterium freudenreichii 
                 
               
               
                   
                 66 
                 459 
                 
                   Rhodococcus erythropolis 
                 
               
               
                   
                 67 
                 460 
                 
                   Rhodococcus fascians 
                 
               
               
                   
                 68 
                 461 
                 
                   Staphylococcus aureus 
                 
               
               
                   
                 69 
                 462 
                 
                   Streptococcus faecalis 
                 
               
               
                   
                 70 
                 463 
                 
                   Streptomyces ambifaciens 
                 
               
               
                   
                 71 
                 464 
                 
                   Streptomyces galbus 
                 
               
               
                   
                 72 
                 465 
                 
                   Streptomyces griseus 
                 
               
               
                   
                 73 
                 466 
                 
                   Streptomyces lividans 
                 
               
               
                   
                 74 
                 467 
                 
                   Streptomyces mashuensis 
                 
               
               
                   
                 75 
                 468 
                 
                   Flavobacterium resinovorum 
                 
               
               
                   
                 76 
                 469 
                 
                   Sphingobacterium multivorans 
                 
               
               
                   
                 77 
                 470 
                 
                   Synechococcus 
                 
               
               
                   
                 78 
                 471 
                 
                   Synechocystis 
                 
               
               
                   
                 79 
                 472 
                 
                   Borrelia burgdorferi 
                 
               
               
                   
                 80 
                 473 
                 
                   Chlamydia trachomatis 
                 
               
               
                   
                 81 
                 474 
                 
                   Azotobacter vinelandii 
                 
               
               
                   
                 82 
                 475 
                 
                   Cowdria ruminantium 
                 
               
               
                   
                 83 
                 476 
                 
                   Mycobacterium intracellulare 
                 
               
               
                   
                 84 
                 477 
                 
                   Mycobacterium lufu 
                 
               
               
                   
                 85 
                 478 
                 
                   Mycobacterium simiae 
                 
               
               
                   
                 86 
                 479 
                 
                   Mycobacterium smegmatis 
                 
               
               
                   
                 87 
                 480 
                 
                   Saccharomonospora azurea 
                 
               
               
                   
                 88 
                 481 
                 
                   Saccharomonospora caesia 
                 
               
               
                   
                 89 
                 482 
                 
                   Saccharomonospora cyanea 
                 
               
               
                   
                 90 
                 483 
                 
                   Saccharomonospora glauca 
                 
               
               
                   
                 91 
                 484 
                 
                   Saccharomonospora viridis 
                 
               
               
                   
                 92 
                 485 
                 
                   Wolbachia pipientis 
                 
               
               
                   
                 93 
                 525 
                 
                   Sphingomonas paucimobilis 
                 
               
               
                   
                 94 
                 526 
                 
                   Zymomonas mobilis 
                 
               
               
                   
                 95 
                 527 
                 
                   Alcaligenes 
                 
               
               
                   
                 96 
                 528 
                 
                   Borrelia burgdorferi 
                 
               
               
                   
                 97 
                 529 
                 
                   Xanthomonas campestris 
                 
               
               
                   
                 98 
                 530 
                 
                   Cowduria ruminantium 
                 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Primers for detection of bacterial species or genera 
               
             
          
           
               
                   
                   
                 SEQ 
                   
                 Reverse primer 
               
               
                 No. 
                 Species used 
                 ID 
                 Forward primer 
                 (reverse primer* = complementary) 
               
               
                   
               
             
          
           
               
                 1 
                 
                   Budvicia aquatica 
                 
                 96 
                 CGAGGTGTTTTAAGGAAAGTT 
                 CGGTCAATAGACAGAATAT 
               
               
                 2 
                 
                   Buttiauxellis agrestis 
                 
                 97 
                 CGAAGGTGTTTTGGTTGAGAG 
                 GGTTGATGAAACAGAATAT 
               
               
                 4 
                 
                   Enterobacter agglomerans 
                 
                 98 
                 CGAAGATGTTTTGGCGGATTG 
                 GTTTCTGGCAACAGAATTT 
               
               
                 5 
                 
                   Erwinia carotovora 
                 
                 99 
                 CGAAGGTGTTTTGAGAGTGAC 
                 TTGGGATGAAACAGAATTT 
               
               
                 6 
                 
                   Erwinia chrysanthemi 
                 
                 100 
                 CGAAGGTGTTTTAGAGAGATT 
                 TCGGGATGAAACAAAATTT 
               
               
                 7 
                 
                   Escherichia coli 
                 
                 101 
                 CGAAGCTGTTTTGGCGGATGA 
                 GTCTGATAAAACAGAATTT 
               
               
                 8 
                 
                   Escherichia hermannii 
                 
                 102 
                 CAGAGTGGTTTTGGTGTTGCG 
                 CAGCAGGTGAACAGAATTT 
               
               
                 9 
                 
                   Escherichia vulneris 
                 
                 103 
                 CGAAGATGTTTTGGCGGATTT 
                 CGTCAGACAGACAGAATTT 
               
               
                 10 
                 
                   Hafnia alvei 
                 
                 104 
                 CGAAGGTGTTTTAAGACGCAG 
                 GGTACAAATAACAGAATAT 
               
               
                 11 
                 
                   Klebsiella oxytoca 
                 
                 105 
                 CGAAGATGTTTTGGCGATTTG 
                 GTTTCTGACAACAGAATTT 
               
               
                 12 
                 
                   Kluyvera cryoescens 
                 
                 106 
                 CAAAGATGTTTTGGTGAAAAG 
                 CGGGTTAATAACAGAATTT 
               
               
                 13 
                 
                   Morganella morganii 
                 
                 107 
                 CGAAGGTGTTTTGAGTTGAGA 
                 TTTGGATTGAAATGAATTT 
               
               
                 14 
                 
                   Pantoea dispersa 
                 
                 108 
                 CAGAGGCGTTTTGGTCTGAGA 
                 GCGGTNTAAAACAAAATTT 
               
               
                 15 
                   Pantoea  ssp. 
                 109 
                 CGAAGATGTTTTGGCGGAATG 
                 GTTTCTGGCAACAGAATTT 
               
               
                 16 
                 
                   Proteus mirabilis 
                 
                 110 
                 CGAAAGTGTTTTGTCAGAGAG 
                 AGTGATTAAAACCGAATTT 
               
               
                 17 
                 
                   Proteus rettgeri 
                 
                 111 
                 CGAAGGTGTTTTAGAGAGATA 
                 CGGGAACAAAACAGAATTT 
               
               
                 18 
                 
                   Providencia stuartii 
                 
                 112 
                 CGAAGGTGTTTTAGAGAGACG 
                 ACGGGAACGAACCGAATTT 
               
               
                 19 
                 
                   Rahnella aquatilis 
                 
                 113 
                 CGAAGGTGTTTTTGATTTGAG 
                 TATGAATGAAACAGAATTT 
               
               
                 20 
                 
                   Salmonella typhi 
                 
                 432 
                 CGAAGGTGTTTTGGAGGATAA 
                 GATAAAAGAAACAGAATTT 
               
               
                 21 
                 
                   Serratia ficaria 
                 
                 114 
                 CGAAGGTGTTTTAGAGAGACG 
                 CAAGAATGAAACAGAATTT 
               
               
                 22 
                 
                   Serratia fonticola 
                 
                 115 
                 CCAAGGTGTTTTGAAGAGATT 
                 TTGAAATGAAACAGAATTT 
               
               
                 23 
                 
                   Serratia marcescens 
                 
                 116 
                 CGAAGGTGTTTTTAGAGAGAT 
                 TTGGAATGAAACAGAATTT 
               
               
                 24 
                 
                   Serratia plymuthica 
                 
                 117 
                 CGAAGGTGTTTTAGAGAGATT 
                 TTGGAATGAAACAGAATTT 
               
               
                 25 
                 
                   Serratia proteamaculans 
                 
                 118 
                 CAAAGGTGTTTTAGAGAGATT 
                 TTGGAATGAAACANAATTT 
               
               
                 26 
                 
                   Serratia rubidea 
                 
                 119 
                 CGAAGGTGTTTTAGAGAGATT 
                 TCGGGATGAAACAGAATTT 
               
               
                 27 
                 
                   Yersinia enterolytica 
                 
                 120 
                 CAAAGGTGTTTTGTATTTGAG 
                 GTTAGTTTAGACAGAATTT 
               
               
                 28 
                 
                   Acinetobacter calcoaceticus 
                 
                 122 
                 CCAAGCAGTTGTATATAAAGC 
                 GCAACCAATAAGACCAATG 
               
               
                 29 
                 
                   Aeromonas enteropelogenes 
                 
                 123 
                 CCAAGAAGTGTTTNTGGTGCT 
                 TTCCAAGATTGAAGATTTT 
               
               
                 30 
                 
                   Aeromonas hydrophila 
                 
                 124 
                 CCAAGAAGTGTTCTAAGGCTT 
                 TTCTCAGATTGAAGAATTT 
               
               
                 31 
                 
                   Buchnera aphidocola 
                 
                 433 
                 CCAGAGGTGTTTTTTATAAAA 
                 ATCTTGTTTTACTGAATTT 
               
               
                 32 
                 
                   Haemophilus influenzae 
                 
                 126 
                 GCTCAAGTGTTTTTGGGAGCT 
                 CGGTCAGTAAACAGAATTT 
               
               
                 33 
                 
                   Moraxella catarrhalis 
                 
                 127 
                 ACCCAAGTGGTTTACCACTGA 
                 GTAATAAACAGACTCATAC 
               
               
                 34 
                 
                   Pasteurella pneumotropica 
                 
                 128 
                 ACCAAATTTGTTTATCGTAAC 
                 AGTTGTTATAATAAAACAT 
               
               
                 35 
                 
                   Vibrio alginolyticus 
                 
                 130 
                 CCAAGGGGTTTTGATGGACTC 
                 TTTCCAGATTAAAGAATTT 
               
               
                 36 
                 
                   Vibrio fisheri 
                 
                 131 
                 CCAAGTGGTTTGTATCAAGCA 
                 TTAAGTAAAACAAACACAG 
               
               
                 37 
                 
                   Vibrio harveyi 
                 
                 132 
                 CCAAGGGGTTTTGATGGACTC 
                 TTTCCAAATTAAAGAATTT 
               
               
                 38 
                 
                   Vibrio parahaemolyticus 
                 
                 133 
                 CCAAGGGGTTTTGATGGACTC 
                 TTTCCGAATTAAAGAATTT 
               
               
                 39 
                 
                   Vibrio proteolyticus 
                 
                 134 
                 CCAAGGGGTTTTGATGGACTC 
                 TTGTTCCAGACAAAATTTT 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Detection potential and specification of the 
               
               
                 location of DNA fragments from the rDNA operon 
               
             
          
           
               
                 No. in 
                   
                 Position in 
                   
               
               
                 FIG. 2 
                 DNA region 
                 SEQ ID 1 
                 Detection potential 
               
               
                   
               
               
                 1. 
                 Terminal region of the 
                 2667–2720 
                 Phyla, classes, orders, 
               
               
                   
                 23 S rDNA gene 
                   
                 families 
               
               
                 2. 
                 Terminal region of the 
                 2727–2776 
                 Phyla, classes, orders, 
               
               
                   
                 23 S rDNA gene 
                   
                 families 
               
               
                 3. 
                 Terminal region of the 
                 2777–2800 
                 Phylas, classes, orders, 
               
               
                   
                 23 S rDNA gene 
                   
                 families 
               
               
                 4. 
                 Terminal region of the 
                 2801–2838 
                 Classes, orders, families 
               
               
                   
                 23 S rDNA gene 
               
               
                 5. 
                 End of the 23 S rDNA 
                 2857–2896 
                 Phyla, classes, orders, 
               
               
                   
                 gene 
                   
                 families 
               
               
                 6. 
                 Beginning of the 
                 2897–2938 
                 Orders, families, genera, 
               
               
                   
                 23 S–5 S transcribed 
                   
                 species, strains 
               
               
                   
                 spacer 
               
               
                 7. 
                 23 S–5 S transcribed 
                 2939–2983 
                 Genera, species, strains 
               
               
                   
                 spacer 
               
               
                 8. 
                 End of the 23 S–5 S 
                 2984–2999 
                 Families, genera, species, 
               
               
                   
                 transcribed spacer 
                   
                 strains 
               
               
                 9. 
                 Beginning of the 5 S 
                 3000–3032 
                 Phyla, classes, orders, 
               
               
                   
                 rDNA gene 
                   
                 families 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Primers from Example 1 
               
             
          
           
               
                   
                   
                 Annealing temperature 
                   
               
               
                 Forward primer 
                 Reverse primer 
                 (° C.) 
                 FIG. 
               
               
                   
               
               
                 SEQ ID 2 
                 SEQ ID 7–22 
                 62 
                 3 
               
               
                 SEQ ID 2 
                 SEQ ID 23–24 
                 62 
                 4 
               
               
                 SEQ ID 2 
                 SEQ ID 25 
                 67 
                 5 
               
               
                 SEQ ID 3–6 
                 SEQ ID 23–24 
                 62 
                 6 
               
               
                 SEQ ID 3–6 
                 SEQ ID 25 
                 67 
                 7 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Consensus PCR for detection of bacteria 
               
             
          
           
               
                   
                   
                 Primer 
                 Primer 
                 Primer 
                 Primer 
                 Primer 
                 Primer 
                 Primer 
                 Primer 
                 Primer 
                 Primer 
               
               
                   
                   
                 A1 
                 B1 
                 C1 
                 D1 
                 E1 
                 F1 
                 G1 
                 H1 
                 B2 
                 A2 
               
               
                 No. 
                 Taxonomic unit 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
               
               
                   
               
             
          
           
               
                 1 
                 
                   Enterobakterien 
                 
                 1 
                 7–22 
                   
                   
                   
                   
                   
                   
                 4 
                 5 
               
               
                 2 
                 
                   Enterobakterien 
                 
                 26 
                 34 
                 42 
                 54 
                 66 
                 78 
                 85 
                   
                   
                 135 
               
               
                 3 
                 
                   Acinetobacter 
                 
                 27 
                 35 
                 43 
                 55 
                 67 
                 79 
               
               
                 4 
                 
                   Aeromonas 
                 
                 28 
                 36 
                 44 
                 56 
                 68 
                 80 
                 87 
                   
                   
                 155 
               
               
                 5 
                 
                   Haemophilus 
                 
                 29 
                 37 
                 45 
                 57 
                 69 
                 81 
               
               
                 6 
                 
                   Moraxella 
                 
                 30 
                 38 
                 46 
                 58 
                 70 
                 82 
               
               
                 7 
                 
                   Pasteurella 
                 
                 31 
                 39 
                 47 
                 59 
               
               
                 8 
                 
                   Stenotrophomonas 
                 
                 32 
                 40 
                 48 
                 60 
                 72 
                   
                 90 
               
               
                 9 
                 
                   Vibrio 
                 
                 33 
                 41 
               
               
                 10 
                 
                   Vibrio alginolyticus 
                 
                   
                   
                 49 
                 61 
                 73 
                   
                 91 
                 130 
                   
                 160 
               
               
                 11 
                 
                   Vibrio fisheri 
                 
                   
                   
                 50 
                 62 
                 74 
                   
                 92 
                 131 
                   
                 161 
               
               
                 12 
                 
                   Vibrio harveyi 
                 
                   
                   
                 51 
                 63 
                 75 
                   
                 93 
                 132 
                   
                 162 
               
               
                 13 
                 
                   Vibrio parahaemolyticus 
                 
                   
                   
                 52 
                 64 
                 76 
                   
                 94 
                 133 
                   
                 163 
               
               
                 14 
                 
                   Vibrio proteolyticus 
                 
                   
                   
                 53 
                 65 
                 77 
                   
                 95 
                 134 
                   
                 163 
               
               
                 15 
                 
                   Pasteurella pneumotropica 
                 
                   
                   
                   
                   
                 71 
                 83 
                   
                 128 
                   
                 158 
               
               
                 16 
                 
                   Acinetobacter calcoaceticus 
                 
                   
                   
                   
                   
                   
                   
                 86 
                 122 
                   
                 154 
               
               
                 17 
                 
                   Haemophilus influenzae 
                 
                   
                   
                   
                   
                   
                   
                 88 
                 126 
                   
                 156 
               
               
                 18 
                 
                   Moraxella catarrhalis 
                 
                   
                   
                   
                   
                   
                   
                 89 
                 127 
                   
                 157 
               
               
                 19 
                 
                   Budvicia aquatica 
                 
                   
                   
                   
                 166 
                   
                   
                   
                 96 
                   
                 135 
               
               
                 20 
                 
                   Buttiauxella agrestis 
                 
                   
                   
                 187 
                 167 
                   
                   
                   
                 97 
                   
                 136 
               
               
                 21 
                 
                   Enterobacter agglomerans 
                 
                   
                   
                 188 
                 168 
                   
                   
                   
                 98 
               
               
                 22 
                 
                   Erwinia carotovora 
                 
                   
                   
                 189 
                 169 
                   
                   
                   
                 99 
               
               
                 23 
                 
                   Erwinia chrysanthemi 
                 
                   
                   
                 190 
                 170 
                   
                   
                   
                 100 
                   
                 138 
               
               
                 24 
                 
                   Escherichia coli 
                 
                   
                   
                 187 
                 171 
                   
                   
                   
                 101 
                   
                 139 
               
               
                 25 
                 
                   Escherichia hermannii 
                 
                   
                   
                 191 
                 172 
                   
                   
                   
                 102 
                   
                 140 
               
               
                 26 
                 
                   Escherichia vulneris 
                 
                   
                   
                 192 
                 173 
                   
                   
                   
                 103, 165 
                   
                 141 
               
               
                 27 
                 
                   Hafnia alvei 
                 
                   
                   
                 193 
                 174 
                   
                   
                   
                 104 
                   
                 142 
               
               
                 28 
                 
                   Klebsiella oxytoca 
                 
                   
                   
                 187 
                 175 
                   
                   
                   
                 105, 165 
                   
                 143 
               
               
                 29 
                 
                   Kluyvera cryoescens 
                 
                   
                   
                 187 
                 175 
                   
                   
                   
                 106 
                   
                 144 
               
               
                 30 
                 
                   Morganella morganii 
                 
                   
                   
                 194 
                 176 
                   
                   
                   
                 107 
                   
                 145 
               
               
                 31 
                 
                   Pantoea dispersa 
                 
                   
                   
                 187 
                 177 
                   
                   
                   
                 108, 165 
                   
                 146 
               
               
                 32 
                 
                   Pantoea 
                 
                   
                   
                 188 
                 178 
                   
                   
                   
                 109, 165 
                   
                 147 
               
               
                 33 
                 
                   Proteus mirabilis 
                 
                   
                   
                 195 
                 179 
                   
                   
                   
                 110 
               
               
                 34 
                 
                   Proteus rettgeri 
                 
                   
                   
                 196 
                 180 
                   
                   
                   
                 111 
                   
                 148 
               
               
                 35 
                 
                   Providencia stuartii 
                 
                   
                   
                 197 
                 181 
                   
                   
                   
                 112 
                   
                 149 
               
               
                 36 
                 
                   Rahnella aquatilis 
                 
                   
                   
                 198 
                 182 
                   
                   
                   
                 113, 164 
                   
                 149 
               
               
                 37 
                 
                   Serratia ficaria 
                 
                   
                   
                   
                   
                   
                   
                   
                 114, 164 
                   
                 150 
               
               
                 38 
                 
                   Serratia fonticola 
                 
                   
                   
                   
                   
                   
                   
                   
                 115, 164 
               
               
                 39 
                 
                   Serratia marcescens 
                 
                   
                   
                   
                   
                   
                   
                   
                 116, 164 
               
               
                 40 
                 
                   Serratia plymuthica 
                 
                   
                   
                   
                   
                   
                   
                   
                 117, 164 
               
               
                 41 
                 
                   Serratia proteamaculans 
                 
                   
                   
                   
                   
                   
                   
                   
                 118, 164 
               
               
                 42 
                 
                   Serratia rubidea 
                 
                   
                   
                   
                   
                   
                   
                   
                 119, 164 
               
               
                 43 
                 
                   Yersinia enterolytica 
                 
                   
                   
                 199 
                 184 
                   
                   
                   
                 120, 164 
                   
                 152 
               
               
                 44 
                 
                   Yersinia pseudotuberculosis 
                 
                   
                   
                 200 
                 185 
                   
                   
                   
                 121, 164 
                   
                 153 
               
               
                 45 
                 
                   Aeromonas enteropelogenes 
                 
                   
                   
                   
                   
                   
                   
                   
                 123 
               
               
                 46 
                 
                   Aeromonas hydrophila 
                 
                   
                   
                   
                   
                   
                   
                   
                 124 
               
               
                 47 
                 
                   Cedecea davisae 
                 
                   
                   
                 201 
                 186 
                   
                   
                   
                 125 
               
               
                 48 
                 
                   Stenotrophomonas multophila 
                 
                   
                   
                   
                   
                   
                   
                   
                 129 
                   
                 159 
               
               
                 49 
                 
                   Enterobacter agglomerans 
                 
                   
                   
                   
                   
                   
                   
                   
                 137, 165 
               
               
                 50 
                 
                   Serratia 
                 
                   
                   
                   
                 183 
                   
                   
                   
                   
                   
                 151 
               
               
                 51 
                 
                   Citrobacter 
                 
                   
                   
                   
                   
                   
                   
                   
                 202, 203 
               
               
                 52 
                 
                   Salmonella 
                 
                   
                   
                   
                   
                   
                   
                 204–210 
               
               
                 53 
                 
                   Pseudomonas stutzeri 
                 
                 213 
                 252 
                 289 
                 326 
                 361 
                 403 
                   
                 434 
                   
                 488 
               
               
                 54 
                 
                   Thiobacillus ferrooxidans 
                 
                 214 
                 253 
                 290 
                 327 
                 362 
                 404 
                   
                 435 
                   
                 489 
               
               
                 55 
                 
                   Agrobacterium vitis 
                 
                 215 
                 254 
                 291 
                 328 
                 363 
                   
                   
                 436 
                   
                 490 
               
               
                 56 
                 
                   Adalia bipunctata 
                 
                 216 
                 255 
                 292 
                 329 
                 364 
                   
                   
                 437 
                   
                 491 
               
               
                 57 
                 
                   Amycolatopsis orientalis 
                 
                 217 
                 256 
                 293 
                 330 
                   
                   
                   
                 438 
               
               
                 58 
                 
                   Brucella ovis 
                 
                 218 
                 257 
                 294 
                 331 
                 365 
                   
                   
                 439 
                   
                 492 
               
               
                 59 
                 
                   Bradyrizobium japonicum 
                 
                 219 
                 258 
                 295 
                 331 
                 366 
                   
                   
                 440 
                   
                 493 
               
               
                 60 
                 
                   Pseudomonas paucimobilis 
                 
                 220 
                 259 
                 296 
                 332 
                 367 
                   
                   
                 441 
                   
                 494 
               
               
                 61 
                 
                   Rhodobacter sphaeroides 
                 
                 221 
                 260 
                 297 
                 333 
                 368 
                   
                   
                 442 
                   
                 495 
               
               
                 62 
                 
                   Rickettsia prowazekii 
                 
                 222 
                 261 
                 298 
                 333 
                 369 
                   
                   
                 443 
                   
                 496 
               
               
                 63 
                 
                   Sphingomonas paucimobilis 
                 
                 223 
                 262 
                 299 
                 334 
                 370 
                 405 
                   
                 525 
                   
                 499 
               
               
                 64 
                 
                   Zymomonas mobilis 
                 
                 224 
                 263 
                 300 
                 335 
                 371 
                   
                   
                 526 
                   
                 500 
               
               
                 65 
                 
                   Alcaligenes 
                 
                 225 
                 264 
                 301 
                 336 
                 372 
                 406 
                   
                 527 
                   
                 501 
               
               
                 66 
                 
                   Pseudomonas cepacia 
                 
                 226 
                 265 
                 302 
                 337 
                   
                 407 
                   
                 444 
                   
                 502 
               
               
                 67 
                 
                   Ralstonia pickettii 
                 
                 227 
                 266 
                 303 
                 338 
                 373 
                 408 
                   
                 445 
                   
                 503 
               
               
                 68 
                 
                   Campylobacter jejuni 
                 
                 228 
                 267 
                 304 
                 339 
                 374 
                 409 
                   
                 446 
               
               
                 69 
                 
                   Helicobacter pylori 
                 
                 229 
                 268 
                 305 
                 340 
                 375 
                 410 
                   
                 447 
                   
                 504 
               
               
                 70 
                 
                   Actinoplanes utahensis 
                 
                 230 
                 269 
                 306 
                 341 
                   
                 411 
                   
                 448 
               
               
                 71 
                 
                   Bacillus halodurans 
                 
                 231 
                 270 
                 307 
                 342 
                 376 
                 412 
                   
                 449 
                   
                 505 
               
               
                 72 
                 
                   Bacillus subtilis 
                 
                 232 
                   
                   
                 343 
                 377 
                 413 
                   
                 450 
                   
                 506 
               
               
                 73 
                 
                   Clostridium tyrobutyricum 
                 
                 233 
                 271 
                 308 
                 344 
                 378 
                 414 
                   
                 451 
                   
                 507 
               
               
                 74 
                 
                   Frankia 
                 
                 234 
                 272 
                 309 
                 345 
                 379 
                 415 
                   
                 452 
                   
                 508 
               
               
                 75 
                 
                   Microbispora bispora 
                 
                 235 
                 273 
                 310 
                 346 
                 380 
                 416 
                   
                 453 
                   
                 509 
               
               
                 76 
                 
                   Mycobacterium leprae 
                 
                 236 
                 274 
                 311 
                 347 
                 381 
                 417 
                   
                 454 
                   
                 510 
               
               
                 77 
                 
                   Mycobacterium smegmatis 
                 
                 237 
                 275 
                 312 
                 348 
                 382 
                 418 
                   
                 455 
                   
                 511 
               
               
                 78 
                 
                   Mycobacterium tuberculosis 
                 
                 238 
                 276 
                 313 
                 349 
                 383 
                 419 
                   
                 456 
                   
                 512 
               
               
                 79 
                 
                   Mycobacterium gallisepticum 
                 
                 239 
                 277 
                 314 
                   
                 384 
                 420 
                   
                 457 
               
               
                 80 
                 
                   Propionibacterium freudenreich 
                 
                 240 
                 278 
                 315 
                 350 
                 385 
                 421 
                   
                 458 
               
               
                 81 
                 
                   Rhodococcus erythropolis 
                 
                 241 
                 279 
                 316 
                 351 
                 386 
                 422 
                   
                 459 
                   
                 513 
               
               
                 82 
                 
                   Rhodococcus fascians 
                 
                 242 
                   
                   
                   
                 387 
                 423 
                   
                 460 
                   
                 514 
               
               
                 83 
                 
                   Staphylococcus aureus 
                 
                 243 
                 280 
                 317 
                 352 
                 388 
                 424 
                   
                 461 
                   
                 515 
               
               
                 84 
                 
                   Streptococcus faecalis 
                 
                 244 
                 281 
                 318 
                 353 
                 389 
                 425 
                   
                 462 
                   
                 516 
               
               
                 85 
                 
                   Streptomyces ambifaciens 
                 
                 245 
                 282 
                 319 
                 354 
                 390 
                 426 
                   
                 463 
                   
                 517 
               
               
                 86 
                 
                   Flavobacterium resinovorum 
                 
                 246 
                 283 
                 320 
                 355 
                 395 
                 428 
                   
                 468 
                   
                 519 
               
               
                 87 
                 
                   Sphingobacterium multivorans 
                 
                 247 
                 284 
                 321 
                 356 
                 396 
                   
                   
                 469 
                   
                 520 
               
               
                 88 
                 
                   Synechococcus 
                 
                 248 
                 285 
                 322 
                 357 
                 397 
                 429 
                   
                 470 
                   
                 521 
               
               
                 89 
                 
                   Synechocystis 
                 
                 249 
                 286 
                 323 
                 358 
                 398 
                 430 
                   
                 471 
                   
                 522 
               
               
                 90 
                 
                   Borrelia burgdorferi 
                 
                 250 
                 287 
                 324 
                 359 
                 399 
                   
                   
                 472, 428 
                   
                 523 
               
               
                 91 
                 
                   Chlamydia trachomatis 
                 
                 251 
                 288 
                 325 
                 360 
                 400 
                 431 
                   
                 473 
                   
                 524 
               
               
                 92 
                 
                   Streptomyces galbus 
                 
                   
                   
                   
                   
                 391 
                 426 
                   
                 464 
               
               
                 93 
                 
                   Streptomyces griseus 
                 
                   
                   
                   
                   
                 392 
                 426 
                   
                 465 
                   
                 518 
               
               
                 94 
                 
                   Streptomyces lividans 
                 
                   
                   
                   
                   
                 393 
                 426 
                   
                 466 
                   
                 518 
               
               
                 95 
                 
                   Streptomyces mashuensis 
                 
                   
                   
                   
                   
                 394 
                 427 
                   
                 467 
               
               
                 96 
                 
                   Salmonella typhi 
                 
                   
                   
                   
                   
                   
                 401 
                   
                 432 
                   
                 486 
               
               
                 97 
                 
                   Buchnera aphidocola 
                 
                   
                   
                   
                   
                   
                   
                   
                 433 
                   
                 487 
               
               
                 98 
                 
                   Brucella orientalis 
                 
                   
                   
                   
                   
                   
                   
                   
                 439 
                   
                 492 
               
               
                 99 
                 
                   Brucella abortus 
                 
                   
                   
                   
                   
                   
                   
                   
                 439 
                   
                 492 
               
               
                 100 
                 
                   Azotobacter vinelandii 
                 
                   
                   
                   
                   
                   
                   
                   
                 474 
               
               
                 101 
                 
                   Cowduria ruminantium 
                 
                   
                   
                   
                   
                   
                   
                   
                 475, 530 
               
               
                 102 
                 
                   Mycobacterium intracellulare 
                 
                   
                   
                   
                   
                   
                   
                   
                 476 
               
               
                 103 
                 
                   Mycobacterium lufu 
                 
                   
                   
                   
                   
                   
                   
                   
                 477 
               
               
                 104 
                 
                   Mycobacterium simiae 
                 
                   
                   
                   
                   
                   
                   
                   
                 478 
               
               
                 105 
                 
                   Mycobacterium smegmatis 
                 
                   
                   
                   
                   
                   
                   
                   
                 479 
               
               
                 106 
                 
                   Saccharomonospora azurea 
                 
                   
                   
                   
                   
                   
                   
                   
                 480 
               
               
                 107 
                 
                   Saccharomonospora caesia 
                 
                   
                   
                   
                   
                   
                   
                   
                 481 
               
               
                 108 
                 
                   Saccharomonospora cyanea 
                 
                   
                   
                   
                   
                   
                   
                   
                 482 
               
               
                 109 
                 
                   Saccharomonospora glauca 
                 
                   
                   
                   
                   
                   
                   
                   
                 483 
               
               
                 110 
                 
                   Saccharomonospora viridis 
                 
                   
                   
                   
                   
                   
                   
                   
                 484 
               
               
                 111 
                 
                   Wolbachia pipientis 
                 
                   
                   
                   
                   
                   
                   
                   
                 485 
               
               
                 112 
                 
                   Rickettsia bellii 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 497 
               
               
                 113 
                 
                   Rickettsia rickettsii 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 498 
               
               
                 114 
                 
                   Xanthomonas campestris 
                 
                   
                   
                   
                   
                   
                   
                   
                 529