MXPA01006838A - Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories - Google Patents

Abstract

The present invention relates to DNA-based methods for universal bacterial detection, for specific detection of the pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epiderminis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis as well as for specific detection of commonly encountered and clinically relevant bacterial antibiotic resistance genes directly from clinical specimens or, alternatively, from a bacterial colony. The above bacterial species can account for as much as 80%of bacterial pathogens isolated in routine microbiology laboratories. The core of this invention consists primarily of the DNA sequences from all species-specific genomic DNA fragments selected by hybridization from genomic libraries or, alternatively, selected from data banks as well as any oligonucleotide sequences derived from these sequences which can be used as probes or amplification primers for PCR or any other nucleic acid amplification methods. This invention also includes DNA sequences from the selected clinically relevant antibiotic resistance genes.

Description

SPECIFIC AND UNIVERSAL PROBES AND PREPARATORS OF AMPLIFICATION TO DETECT AND IDENTIFY QUICKLY COMMON BACTERIAL PATHOGENIC AGENTS AND GENES RESISTANT TO ANTIBIOTICS FROM SPECIMENS CLINICS FOR HABITUAL DIAGNOSIS IN LABORATORIES D MICROBIOLOGY BACKGROUND OF THE INVENTION Classical Identification of Bacteria Bacteria are classically identified by their ability to utilize different substrates as a carbon and nitrogen source, through the use of biochemical tests, such as the API20E ™ system. Tests for susceptibility to Gram-negative bacilli have progressed to microdilution tests. Although API and microdilution systems are cost-effective, at least two days are required to obtain preliminary results, due to the need for two successive incubations during the noch to isolate and identify specimen bacteria. It has developed some more rapid detection methods with sophisticated and expensive devices. For example, the fastest identification system, the autoSCAN-Walk-Away ™ system, identifies both Gram-negative and Gram-positive bacteria from isolated bacterial colonies in 2 hours, and patterns of antibiotic susceptibility in only 7 hours . However, this system has an unacceptable margin of error, especially with bacterial species different from Enterobacteriaceae (York et al., 1992. J. Clin Microbiol 302903-2910). However, even this fastest method requires primary isolation of the bacteria with a pure culture, a process which takes at least 1 hour if there is a pure culture or 2 to 3 days if there is a mixed culture.

Urine Specimens A large proportion (40-50%) of the specimens received in the usual diagnostic microbiology laboratories for the identification of bacteria are the urine specimens (Pezzlo, 1988, Clin.Microbiol Rev. 1: 268- 280). Urinary tract infections (UT) are extremely common and affect up to 20% of women and add excessive morbidity and increased mortality among hospitalized patients (Johnson and Stamm, 1989, Ann Intern Med. 111: 906- 917). UTIs are usually bacterial etiology and require antimicrobial therapy. The Gram negative bacillus Escherichia coli is by far the most prevalent pathogen of urine and adds up to 50 to 60 of the ICUs (Pezzlo, 1988, op.cit.). The occurrence of bacterial pathogens isolated from specimens of the orin recently observed in the "Center Hospitalier d l" Université Laval (CHUL) "is given in Tables 1 and 2.

Conventional Identification of Pathogens in Urine Specimens. The search for pathogens in urine specimens is thus preponderant in the usual microbiology laboratory, that innumerable tests have been developed. The Gold standard is still a semi-quantitative classical plaque culture method, in which a urine cycle is placed in strips and incubated for 18 to 24 hours. The colonies are then counted to determine the total number of units that form colonies (CFU) per liter of urine. A UTI is usually associated with a bacterial account of > 107 CFU / 1 in the urine. However, infections with less than 107 CFU / 1 of urine are possible, particularly in patients with high incidence of diseases or those catheterized (Stark and Maki, 1984, N. Engl. "Med. 311: 560-564).

Importantly, about 80%. of tested urine specimens are considered negative (<107 CFU / 1, Table 3). Accurate and rapid urine classification methods for bacterial pathogens would allow faster identification of negative results and more efficient clinical investigation. Several rapid identification methods (Uriscreen ™, UTIscreen ™, Flash Track ™ DNA examination and others) were recently compared to the slower standard biochemical methods, which are based on the cultivation of bacterial pathogens. Although they are much faster, these rapid tests showed low sensitivities and specificities as well as a high number of false negative and positive results (Koening et al., 1992, "Clin. Microbiol. 30: 342-345.; Pezzlo et al. , 1992, J. Cli Microbiol. 30: 640-684). Urine specimens found positive for cultures are further characterized using standard biochemical tests to identify < the • bacterial pathogen was also tested for susceptibility to antibiotics.

Any Clinical Specimen As with the urine specimen, which was used here as an example, our examinations and preparation (primers) of the amplification are also applicable to any other clinical specimen. The DNA-based tests proposed in this invention are superior to the standard methods currently used for routine diagnostics in terms of speed and accuracy. While a high percentage of urine specimens are negative, and many other clinical specimens more than 95% of the cultures are negative (Table 4). These data also support the use of universal tests to classify clinical negative specimens. Clinical specimens of organisms in addition to humans ^ (for example of primates, mammals, farm animals or livestock) can also be used.

Towards the • development of rapid DNA-based diagnostic tests A rapid diagnostic test should have a significant impact on the management of infections. For the identification of pathogens and antibiotic-resistant genes in clinical samples, DNA testing and DNA amplification technologies offer several advantages over conventional methods. There is no need for sub-cultures, here the organism can be directly detected in. the clinical samples, thus reducing the costs and time associated with the isolation of pathogens. DNA-based technologies have proven to be extremely useful for specific applications in the clinical microbiology laboratory. For example, equipment for the detection of organisms based on the use of hybridisation probes or the amplification of DNA for the direct detection of pathogens in clinical specimens, is commercially available (Persing et al., 1993, Diagnosti Molecular Microbiology: Principies and Applications, America Society for Microbiology, Washington, D. C). The present invention is an advantageous alternative to conventional methods of crop identification, used in clinical microbiology laboratories of hospitals, and in private clinics for routine diagnosis. In addition, being much faster, diagnostic tests based on DNA are more accurate than the standard biochemical tests currently used for diagnosis, because the bacterial genotype (for example, the DNA level) is more stable than the bacterial phenotype. (for example, the biochemical properties). The originality of this invention is that the genomic DNA fragments (size of at least 100 base pairs) specific for 12 bacterial pathogen species commonly found are selected from genomic libraries or from data banks. Amplification primers or oligonucleotide probes (both less than 100 nucleotides in length), which are both derived from the sequence of species-specific DNA fragments, identified by the hybridization of genomic libraries or selected sequences from data banks are used as a basis for developing diagnostic tests. Oligonucleotide primers and probes for the detection of bacterial resistance genes commonly found and clinically important are also included. For example, the present Annexes I and II present a list of suitable oligonucleotide probes and PCR primers, which are all derived from DNA fragments of specific species, selected from genomic libraries or from databank sequences. It is clear to a person skilled in the art that sequences of oligonucleotides appropriate for the specific detection of the above bacterial species, in addition to those listed in Annexes 1 and 2, can be delivered from fragments of specific species or sequences of Selected data banks. For example, the oligonucleotides may be shorter or longer than those we have chosen and may be selected from any site in the identified sequences of specific species or sequences of selected data banks. Alternatively, oligonucleotides can be designed for use in amplification methods in addition to PCR. Consequently, the core of this invention is the identification of genomic DNA fragments of specific species from genomic DNA libraries and the selection of genomic DNA fragments from databank sequences, which are used as a source of oligonucleotides. from. specific and ubiquitous species. Although the selection of oligonucleotides suitable for the purpose of diagnosing the sequence of fragments of specific species or of the sequences selected from the database requires a lot of effort, it is very possible for a person skilled in the art to derive from our fragments or from sequences selected from "adequate" oligonucleotide data banks that are different from those we have selected and tested as examples (Annexes I and II).

Others have developed DNA-based tests for the detection and identification of some bacterial pathogens (PCT patent application, Serial No. WO 93/03186). However, its strategy is based on the • 5 amplification of the highly conserved 16S rRNA gene, followed by hybridization with internal oligonucleotides of specific species. The strategy of this invention is much simpler and faster, because it allows the direct amplification of the targets of specific species using the oligonucleotides derived from the bacterial genomic DNA fragments of specific species. Since a high percentage of clinical specimens are negative, oligonucleotide primers and probes were selected from the highly conserved 16S or 23S, 15 rRNA genes, to detect all bacterial pathogens possibly found in clinical specimens, in order to determine if a clinical specimen is infected or not. This strategy allows the rapid classification of the numerous negative clinical specimens, • 20 submitted for bacteriological tests. We have also developed other tests based on DNA, to be performed simultaneously with bacterial identification, to quickly determine the presumed bacterial susceptibility to antibiotics, commonly targeting genes that are resistant to bacteria found and clinically relevant. Although sequences from selected genes resistant to antibiotics are available and have been used to develop DNA-based tests for their detection (Ehrlich and Greenber, 1994, PCR-based Diagnostics in Infectious Diseases, Blackwell Scientific Publications, Boston, Massachusetts; Persing et al. , 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D. < "•), our approach is innovative, as it represents improvements important on current diagnostic methods "Gold ? ^ standard ", based on the cultivation of bacteria, because it allows the rapid identification of the presence of a specific bacterial pathogen and the evaluation of its susceptibility to antibiotics directly from the clinical specimens within one hour. We believe that rapid and simple diagnostic tests, not based on the cultivation of bacteria, that we are developing, will gradually replace the conventional, used bacterial identification methods. currently in clinical microbiology laboratories of hospitals and in private clinics. In our opinion, these rapid diagnostic tests based on DNA for severe and common bacterial agents and antibiotic resistance (i) will save lives for the perfection of treatment, (ii) will reduce antibiotic resistance, reduce the use of broad-spectrum antibiotics, and (iii) lower overall health costs by preventing or shortening hospitalizations.

COMPENDIUM OF THE INVENTION In accordance with the present invention, a sequence of genomic DNA fragments (size of at least 100 basc and all pairs described in the sequence list) selected or by hybridization of genomic or bank libraries is provided. of data and which are specific for the detection of commonly found bacterial pathogens (ie, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis) in clinical specimens. These bacterial species are associated with approximately 90% of urinary tract infections and with a high percentage of other severe infections, including septicemia, meningitis, pneumonia, intra-abdominal infections, skin infections and many other severe infections of the respiratory tract. In general, the above bacterial species can add up to 80% of the bacterial pathogens isolated in the usual microbiology laboratories. Synthetic oligonucleotides for hybridization (probes) or DNA amplification (primers) are derived from the above DNA fragments of specific species (ranging in size from 0.25 to 5.0 kilobase pairs (kbp) or selected bank sequences). of data (GenBank and EMBL) The bacterial species for which some of the oligonucleotide probes and amplification primers are derived from sequences of selected data banks are: Escherichia coli, Enterococcus f ecalis, Streptococcus pyogenes and Pseudomonas aeruginosa. Those skilled in the art will understand that the important innovation in this invention is the identification of DNA fragments of specific species, selected from bacterial genomic libraries by hybridization or from databank sequences.The selection of oligonucleotides from these fragments suitable for diagnostic purposes is also innovative. Specified and ubiquitous leotides, different from those tested in practice, are considered as embodiments of the present invention. The development of the hybridization (with the fragment or probes of the oligonucleotide) or of the DNA amplification protocols for the detection of pathogens of the clinical specimens, make possible a very rapid bacterial identification. This will greatly reduce the time currently required for the identification of pathogens in the clinical laboratory, since these 5 technologies can be applied for bacterial detection and identification directly from clinical specimens with minimal prior treatment of any biological specimen to release the DNA bacterial. In addition to being 100% specific, amplification probes and primers allow the identification of bacterial species directly from clinical specimens or, alternatively, from an isolated colony. DNA amplification assays have the additional advantages of being faster and more sensitive than hybridization assays, since they allow the rapid and exponential in vitro reproduction of the target segment of the DNA from the bacterial genome. Probes or universal exams and selected amplification primers of the 16S or 23S rRNA genes highly conserved among the bacteria, which allow the The detection of any bacterial pathogen will serve as a procedure for classifying the numerous negative clinical specimens received in diagnostic laboratories. The use of oligonucleotide probes or complementary preparations. to the characterized bacterial genes which encode resistance to antibiotics, to identify the commonly found and clinically important resistant genes is also considered to be within the scope of this invention.

DETAILED DESCRIPTION OF THE INVENTION Development of DNA Probes of Specific Species Probes of DNA fragments were developed for the following bacterial species: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermis, Staphylococcus saprophyticus, • Haemophilus influenzae and Moraxella catarrhalis. (For Enterococcus faecalis and Streptococcus pyogenes, the oligonucleotide sequences are derived exclusively from the selected sequences of data banks). These fragments of specific species were selected from bacterial genomic libraries by DNA hybridization of a variety of Gram positive and Gram negative bacteria species (Table 5). • 20 The chromosomal DNA of each bacterial species for which the probes were searched were isolated using methods .standard. The DNA was digested with a restriction enzyme frequently cut, such as Sau3AI, and then ligated into the bacterial plasmid vector pGEM3Zf (Promega), linearized by the appropriate digestion of the restriction endonuclease. The recombinant plasmids were then used to transform the competent E. coli strain DH5a, thus providing a genomic library. The plasmid content of the transformed bacterial cells was analyzed using standard methods. The DNA fragments of target bacteria vary in size from 0.25 to 5.0 kilobase pairs (kbp), were cut from the vector by the digestion of the recombinant plasmid with several restriction endonucleases. The insert was separated from the vector by the gel electrophoresis of agarose and purified on low-melting agarose gels. Each purified fragment of bacterial genomic DNA • It was then used as a probe for specificity tests. For each given species, gel-purified restriction fragments of an unknown encoder potential were labeled with the radioactive nucleotide a-32P (dATP), which was incorporated into the DNA fragment by the randomization labeling reaction. Non-radioactive modified nucleotides can also be incorporated into the DNA by this method to serve as a label. • Each probe of the DNA fragment (ie, a segment of bacterial genomic DNA less than 100 bp in length, cut out from clones randomly selected from the genomic library) was then tested in its specificity by hybridization to the DNA of a variety of species bacterial (Table 5). The labeled, double-stranded DNA probe was heat denatured to deliver a single-lane labeled DNA, which then hybridizes to any single-strand target DNA, fixed to a solid or solid solution support. The target DNAs consist of DNA total cell of an array of bacterial species found in clinical samples (Table 5). Each target DNA was released from the bacterial cells and denatured by conventional methods and then fixed irreversibly on a solid support (eg nylon or nylon membranes). nitrocellulose) or free in solution. The fixed single-stranded target DNAs were then hybridized with a single-stranded probe. The conditions of pre-hybridization, hybridization and post-hybridization were as follows: (i) pre-hybridization in 1 M NaCl + 10% dextran sulfate + 1% SDS (sodium dodecyl sulfate) + 100 μg / ml of salmon sperm DNA, at 65 ° C for 15 minutes: (ii) hybridization; in a fresh pre-hybridization solution containing the labeled probe, at 65 ° C overnight; ^^ (iii) post-hybridization; washed twice in 3 x SSC that contains 1% SDS (IX SSC is 0.15M NaCl, 0.015M NaCitrate) and twice in 0.1 x SSC containing 0.1% SDS; all washes were at 652C, for 15 minutes. The autoradiography of the washed filters allowed the detection of the selectively hybridized probes. Hybridization of The probe to a specific target DNA indicated a high degree of similarity between the nucleotide sequence of these two DNAs. DNA fragments of specific species, selected from the various bacterial genomic libraries, ranging in size from 0.25 to 5.0 kbp, were isolated for 10 common bacterial pathogens (Table 6), based on hybridization to chromosomal DNA. of a variety of bacteria, performed as described above. All bacterial species tested (66 species listed in Table 5) were probably pathogenic, associated with common infections or potential contaminants, which can be isolated from clinical specimens. A probe of the DNA fragment was considered specific only when it hybridized only to the pathogenic agent from which it was isolated. Probes of DNA fragments, which were found to be specific, were subsequently tested in their ubiquity (ie, the ubiquitous probes recognized more isolated from the target species) by hybridization to the bacterial DNAs of approximately 10 to 80 clinical isolates of the species of interest (Table 6). The DNAs were denatured, fixed on nylon membranes and hybridized as described above.

Sequence of Fragments Probes of Specific Species The nucleotide sequence of all or a portion of the DNA fragments of specific, isolated species (Table 6) was determined using the dideoxynucleotide termination sequence method, which was performed using Sequenasa (USB Biochemicals) or T7 DNA polymerase (Pharmacia). These nucleotide sequences are shown in the list of sequences. Alternatively, selected sequences from the data banks (GenBank and EMBL) were used as sources of oligonucleotides for diagnostic purposes for Escherichia coli, Enterococcus faecalis, Streptococcus pyogenes and Pseudomonas aeruginosa. For this strategy, an array of suitable oligonucleotide primers or probes, derived from a variety of genomic DNA fragments (size greater than 100 bp) selected from data banks, was tested for its specificity and ubiquity in PCR and assays. of hybridization, as described below. It is important to note that the sequences of data banks were selected based on their potential to be of specific species, according to available sequence information. Only sequences of data banks from which oligonucleotides of specific species can be derived are included in this invention. Oligonucleotide probes and amplification primers derived from fragments of specific species, selected from genomic libraries or from databank sequences, were synthesized using an automatic DNA synthesizer (Millipore). Prior to the synthesis, all oligonucleotides (probes for hybridization and primers for DNA amplification) were evaluated in their proper form for hybridization or DNA amplification by the polymerase chain reaction (PCR) by the analysis of computer using standard programs (for example, Genetic Computer Group (GCG) and Oligo ™ 4.0 (National Biosciences)). The appropriate potential form of the PCR preparer pairs was also evaluated before synthesis, verifying the absence of unwanted characteristics, such as the long extensions of a nuleotide, a high proportion of the residues of G or C in the 3 'end and a T residue of terminal 3 • (Persing et al., 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D. c.).

Hybridization with Oligonucleotide Probes In hybridization experiments, oligo-nucleotides (size less than 100 nucleotides) have some advantages over probes of the DNA fragment for the detection of bacteria, such as ease of preparation in large quantities, consistency, consistency in results from one batch to another and chemical stability. Briefly, for the hybridizations, the oligonucleotides are labeled at the 5 'end with the? 32P (ATP) radionucleotide, using the T4 polynucleotide kinase (Pharmacia). The unincorporated radionucleotide was removed by passing the single-lane labeled oligonucleotide through a Sephadex G50 column. Alternatively, the oligonucleotides were labeled with biotin, either enzymatically at their 3 'ends or directly incorporated during synthesis at their 5' ends, or with digoxigenin. It will be appreciated by the person skilled in the art that inscription elements, in addition to the three previous labels, can be used. The target DNA was denatured, fixed on a solid support and hybridized, as previously described for the probes of the DNA fragment. The conditions for pre-hybridization and hybridization are as described above. The post-hybridization washing conditions are as follows: twice in 3X SSC containing 1% SDS, twice in 2X SSC containing 1% SDS and twice in IX SSC containing 1% SDS (all these washes are at 652C for 15 minutes) and a final wash in 0.1X SSC containing 1% SDS at 252C for 15 minutes. For probes labeled with radioactive labels, the detection of the hybrids was by autoradiography, as described above. For non-radioactive labels, the detection can be colorimetric or by chemiluminescence. Oligonucleotide probes can be derived from any double DNA strand. These probes may consist of bases A, G, C or T or the like. The probes can be of any suitable length and can be selected anywhere within the fragments • Genomics of specific species, selected from genomic libraries or from databank sequences.

Amplification of DNA For the amplification of DNA by the PCR method (polymerase chain reaction), widely used, 10 pairs of preparators are derived. or of the DNA fragments in sequence, of specific species, or of sequences • from the data bank or alternatively, shortened versions of the oligonucleotide probes. Prior to synthesis, the potential partners of the preparer were analyzed using the Oligo ™ 4.0 (National Biosciences) program to verify that they are likely candidates for PCR amplifications. During DNA amplification by PCR, two oligonucleotide primers, which bind respectively to each strand of the target DNA, denatured, double • - cord, from the bacterial genome, were used to exponentially amplify the target DNA in vitro by successive thermal cycles that allow DNA denaturation. tempers of the preparers and synthesis of the new objectives in each cycle (Persing et al., 1933. Diagnostic Molecular Microbiology: Principies and Applications, American Society for Microbiology, Washington, D.C.). In short, the PCR protocols are as follows. Clinical specimens or bacterial colonies were added directly to 50 μl of the reaction mixtures containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2, 0.4 μM of each of the two preparators , 200 μM of each of the four dNTPs and 1.25 units of the Taq DNA polymerase (Perkin Elmer). The PCR reactions were then subjected to a thermal cycle (3 minutes at 952C, followed by 30 cycles of 1 second at 952C and 1 second at 55ac), using a Perkin Elmer 480 ™ thermal cycle device and subsequently analyzed by electrophoresis ethidium bromide standard - stained agarose gel. It is clear that other methods of detection of specific amplification products, which can be faster and more practical for the usual diagnosis, can be used. These methods can be based on the detection of fluorescence after amplification (for example the TaqMan ™ system by Perkin ELmer or the Amplensor ™ of Biotronics) or liquid hybridization with an oligonucleotide probe that binds to internal sequences of the specific product of amplification. These novel probes can be generated from our probes of fragments of specific species. ' The methods based on the detection of fluorescence are particularly promising for use in the usual diagnosis, since they are very fast and quantitative and can be made automatic. To ensure the efficiency of the PCR, glycerol or dimethyl sulfoxide (DMSO) or other related solvents can be used to increase the sensitivity of the PCR and overcome the problems associated with the amplification of the target with a high GC content or with strong secondary structures. The concentration ranges for glycerol and DMSO are 5-15% (v / v) and 3-10% (v \ v), respectively. For the PCR reaction mixture, the concentration varies for the amplification preparers and gCl2 are 0.1-1.0 μM and 1.5-3.5 mM, respectively. Modifications of the standard PCR protocol using external and nested primers (ie nested PCR) or using more than one pair of primers (ie multiplex PCR) can also be used (Persing et al., 1993. Diagnostic Molecular Microbiology: Principies and Applications, American Society for Microbiology, Washington. D.C.). For more details about PCR protocols and amplification detection methods, see Examples 7 and 8. Those skilled in the art of amplification are aware of other rapid amplification methods, such as the chain reaction. of ligase (LRC), transcription-based amplification systems (TAS), self-sustained sequence reproduction (3SR), nucleic acid sequence-based amplification (NASBA), cord displacement amplification (SDA) and branched DNA (bDNA) (Persing et al., 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, DC). The scope of this invention is not limited to the use of PCR amplification, but rather includes the use of any rapid method of nucleic acid amplification or any other method that can be used to increase the speed and sensitivity of the tests. Any oligonucleotide suitable for, the amplification of the nucleic acid by other approaches in addition to the PCR and derivatives of fragments of specific species and the sequences of genes with selected antibiotic resistance, included herein, are also within the scope of this invention.

Ubiquitous and Specificity Tests for Oligonusleotide and Preparer Probes The specificity of oligonucleotide probes, derived from fragments in sequence of specific species or from databank sequences, were tested by hybridization to the DNAs of the array of bacterial species listed in Table 5, as previously described. The oligonucleotides that were found to be specific were subsequently tested in their ubiquity by hybridization to the bacterial DNAs of about 80 isolates of the target species, as described for the fragment probes. The probes are considered ubiquitous when they hybridize specifically with the DNA of at least 80% of the isolates. The results for the specificity and ubiquity tests with the oligonucleotide probes are summarized in Table 6. The specificity and ubiquity of the amplification primer pairs were tested directly from cultures (see Example 7) of the same bacterial strains. For specificity and ubiquity tests, PCR assays were performed directly from bacterial colonies of approximately 80 isolates of the target species. The results are summarized in Table 7. All specific and ubiquitous oligonucleotide probes and amplification primers for each of the 12 bacterial species investigated are listed in Annexes I and II, respectively. Divergence in DNA fragments in sequence can occur and, as long as this divergence of these sequences or part of them does not affect the specificity of the probes or the amplification primers, the bacterial DNA variant is within the scope of this invention .

Universal Bacterial Detection In the usual microbiology laboratory, a high percentage of clinical specimens sent for bacterial identification is negative (Table 4). For example, in a period of 2 years, about 80% of urine specimens received in the laboratory, "between Hospitalier de dfc l'Université Laval (CHUL)" were negative (ie <107 5 CFU / 1) (Table 3). Clinical test samples with universal probes or universal amplification primers, to detect the presence of bacteria before the specific identification and classification of the numerous negative specimens is thus useful, as it saves costs and can guide The clinical management of the patients, several oligonucleotides and amplification primers, were rapidly synthesized from highly conserved portions of the bacterial 16S or 23S ribosomal RNA gene sequences, available in data banks (Annexes II and IV). ).

In hybridization tests, a set of seven oligonucleotides (Annex I, Table 6) hybridized strongly to the DNA of all the bacterial species listed in Table 5. This set of universal probes labeled with radionuclides or with any other modified nucleotides is consequently very useful for the detection of bacteria in urine samples with a sensitivity range of > 107 CFU / 1. These probes can also be applied for bacterial detection in other clinical samples. The amplification preparers, too derived from the sequence of highly conserved ribosomal RNA genes were used as an alternative strategy for universal bacterial detection directly from clinical specimens (Annex IV, Table 7). The strategy of DNA amplification was deped to increase the sensitivity and speed of the test. This amplification test is ubiquitous, since it specifically amplifies the DNA of 23 different bacterial species found in clinical specimens. Well-preserved bacterial genes, in addition to the ribosomal RNA genes, can also be good candidates for universal bacterial detection directly from clinical specimens. These genes may be associated with processes essential for bacterial survival (eg, protein synthesis, DNA synthesis, cell division or DNA repair) and may, therefore, be highly conserved during evolution. We are working on these candidate genes to dep new rapid tests for the universal detection of bacteria directly from clinical specimens.

Antibiotic Resistant Genes Antimicrobial resistance complicates treatment and often leads to therapeutic failures. Likewise, the excessive use of antibiotics inevitably leads to the emergence of bacterial resistance. Our goal is to provide the clinics, within an hour, with the necessary information to prescribe the optimal treatments. In addition, rapid identification of negative clinical specimens with DNA-based tests for universal bacterial detection and identification of the • 5 presence of a specific pathogen in specimens positive with DNA-based tests for specific bacterial detection, clinics also need timely information about the ability of the bacterial pathogen to resist antibiotic treatments.

We believe that the most efficient strategy to evaluate bacterial resistance quickly to agents against microbes is to detect directly from clinical specimens the most common and important antibiotic resistance genes (ie, DNA-based tests for detection of antibiotic-resistant genes). Since the sequence of the most important and common bacterial genes with resistance to antibiotics is available from the data banks, our strategy is to use this sequence of a portion or of the whole gene to design oligonucleotides • 20 specific ones that will be used as a basis for the depment of rapid tests based on DNA. The sequence of bacterial genes with antibiotic resistance selected on the basis of their clinical significance (ie high incidence and importance) is given in the sequence listing.

Table 8 summarizes some characteristics of the selected genes resistant to antibiotics.

EXAMPLES • 5 The following examples are intended to be illustrative "of the various methods and compounds of the invention.

EXAMPLE 1: Isolation and Cloning of Fragments 10 The genomic DNAs of: Escherichia coli, strain ATCC 25922, Klebsiella pneumoniae, strain CK2, Pseudomonas aeruginosa, strain ATCC 27853, Proteus mirabilis, strain ATCC 35657, Streptococcus pneumoniae, strain ATCC 27336, Staphylococcus aureus, strain ATCC 25923, Staphylococcus epidermis, strain ATCC 12228, Staphylococcus saprophyticus, strain ATCC 15305, Haemophilus influenzae, reference strain Rd and Moraxella catarrhalis, strain ATCC 53879, were prepared using standard procedures. It will be understood that bacterial genomic DNA may have been isolated from other strains of • 20 those mentioned above. (For Enterococcus faecalis and Streptococcus pyogenes, the oligonucleotide sequences are derived exclusively from data banks). Each DNA was digested with a restriction enzyme, which frequently cuts DNA, such as Sau3AI. The fragments The resulting DNA was ligated into a plasmid vector (pGEM3Zf) to create recombinant plasmids and transformed into the competent cells of E. coli (DH5a). It will be understood that the corresponding competent vectors and cells are not limited to those specifically exemplified hereinbefore. The goal of obtaining recombinant plasmids and transformed cells is to provide an easily reproducible source of DNA fragments useful as probes. Therefore, as long as the inserted fragments are specific and selective for the target bacterial DNA, any recombinant plasmid and the corresponding transformed host cells are within the scope of this invention. The plasmid content of the transformed bacterial cells was analyzed using standard methods. The fragments of the AD of target bacteria, which vary in size from 0.25 to 5.0 kbp, were cut from the vector by digestion of the plasmid resorbated with several restriction endo-nucleases. The insert was separated from the vector by agarose gel electrophoresis and purified on low melting agarose gel. Each of the purified fragments was then used for specificity tests.

Labeling of DNA Fragment Probes. The label used was a32P (dATP), a radioactive nucleotide ^ which can be enzymatically incorporated into a double-stranded DNA molecule. The fragment of interest is first denatured by heating at 95 ° C for 5 minutes, then a mixture of random primers is allowed to harden the strands of the fragments. These primers, once tempered, provide a starting point for the synthesis of DNA. The DNA polymerase, usually the Klenow fragment, is provided together with the # four nucleotides, one of which is radioactive. When the reaction is finished, the mixture of the new DNA molecules is once again denatured to deliver radioactive molecules of double-stranded DNA (ie, the probe). As mentioned before, other nucleotides can be used modified to label the probes.

Specificity and Ubiquity Tests for DNA Fragment Probes DNA fragments of specific species, which range in size from 0.25 to 5.0 kbp, were isolated for 10 common bacterial pathogens (Table 6), based on hybridization to the chromosomal DNA of a variety of bacteria. Examples of all cellular DNA for each bacterial strain, listed in Table 5, were transferred onto a nylon membrane using a spot spot apparatus, washed and denatured before irreversibly fixed. Hybridization conditions are as described above. A DNA fragment probe is considered specific only when it hybridizes only to the pathogen of which was isolated. The labeled fragments of DNA that specifically hybridize only to the target (ie, specific) bacterial species were then probed in their ubiquity by hybridization to the DNAs of approximately 10 to 80 isolates of the species of interest, as described above. The conditions of the pre-hybridization, hybridization and post-hybridization washes are as described above. After autoradiography (or other appropriate detection elements for the non-radioactive label used), the specificity of each individual probe can be determined. Each probe that was found to be specific (ie hybridizes only to the DNA from the bacterial species from which it "was isolated") and ubiquitous (ie, hybridizes to most isolates of the target species), was retained for further experiments.

EXAMPLE 2: Same as Example 1, except that the test of the strains is by colony hybridization. The bacterial strains were inoculated on a nylon membrane, placed on nutrient agar. The membranes were incubated at 37 ° C for two hours and then bacterial lysis and DNA denaturation were carried out according to standard procedures. The DNA hybridization was carried out as described above.

EXAMPLE 3 Same as Example 1, except that the bacteria were detected directly from the clinical samples.

• Any biological sample was loaded directly onto a spot spot apparatus and the cells were subjected to in situ lysis for bacterial detection. Blood samples must be heparinized in order to avoid coagulation that interferes with their convenient loading in the spot spot apparatus.

EXAMPLE 4: Nucleotide Sequences of DNA Fragments. The nucleotide sequence of the whole or a portion of each fragment found., Specific and ubiquitous (Example 1) was determined using the dideoxynucleotide termination sequence method (Sanger et al., 1977, Proc. Nati Acad. Sci. USES. 74: 5463-5467). These DNA sequences are shown in the sequence listing. The probes of Oligonucleotides and amplification primers were selected from these nucleotide sequences or alternatively from sequences from selected data banks and then synthesized on an automated bioinvestigation synthesizer (Millipore ™) using the chemistry of the phosphoramidite.

Oligonucleotide labeling. Each oligonucleotide was labeled at the 5 'end with? 32 P-ATP by the T4 polynucleotide kinase (Pharmacia), as described above. The label can also be non-radioactive.

Specificity Test for Oligonucleotide Probes All oligonucleotide probes were tested in their specificity by hybridization to the DNAs of a variety of Gram-positive and Gram-negative bacteria species, as described above. The probes of specific species were those that hybridize only to the DNA of the bacterial species from which they were isolated. Oligo-nucleotide probes that were found to be specific were submitted to the ubiquity tests as follows.

Ubiquity test for oligonucleotide probes. Then specific probes of oligo-nucleotides were used in the ubiquity tests with approximately 80 strains of the target species. The chromosomal DNAs of the isolates were transferred onto nylon membranes and hybridized with labeled oligonucleotide probes, as described for the specificity tests. The bacteria of approximately 80 isolates, constructed for each target species, contain the reference strains of the ATCC, as well as a variety of clinical isolates, obtained from the various sources. Ubiquitous probes are those that hybridize at least 80% of the DNA of the battery of clinical isolates of the target species. Examples of specific and ubiquitous oligonucleotide probes are listed • in Annex 1.

EXAMPLE 5: Same as Example 4, except that a set of specific oligonucleotide probes is used for bacterial identification (i) to increase sensitivity and ensure 100% ubiquity or (ii) to identify • simultaneously more than one bacterial species. Bacterial identification can be done from isolated colonies or directly from clinical specimens. EXAMPLE 6: PCR amplification The PCR technique was used to increase the sensitivity and rapidity of the tests. The PCR preparers used are often shorter derivatives of extensive sets of • Oligonucleotides previously developed for hybridization assays (Table 6). The sets of primers were tested in PCR assays performed directly from a bacterial colony or from a bacterial suspension (see Example 7) to determine its specificity and ubiquity (Table 7). Examples of pairs of PCR preparers, specific and ubiquitous, are listed in Annex II.

Specificity and Ubiquity Tests for Amplification Preparers The specificity of all selected preparator pairs of the PCR was tested against the battery of Gram-negative and Gram-positive bacteria, used to test the oligonucleotide probes (Table 5). The couples of prepared, specific specifiers for each species were then tested in their ubiquity to ensure that each set • Preparers can amplify at least 80% of the DNA of one. Battery of approximately 80 isolates of the target species. The isolated batteries built from each species contain reference strains of the ATCC and several clinical isolates representative of the clinical diversity for each species. Standard precautions will be taken to avoid false positive PCR results. Methods to inactivate the • 20 PCR amplification products, such as inactivation by uracil-N-glycosylase can be used to control the remaining PCR.

Example 7: 25 Direct Amplification of a Bacterial Colony or Suspension Trials were performed either directly from a bacterial colony or from a bacterial suspension, the latter being adjusted to a standard McFarland of 0.5 (corresponds to 1.5 x 108 bacteria / ml). In the case of the direct amplification of a colony, a portion of the colony was transferred directly to 50 μl of a PCR reaction mixture (containing 50 mM KCl, 10 mM Tris, pH 8.3, 2.5 mM MgCl2 0.4 μM of each of the two preparators, 200 μM of each of the four dNTPs and 1.25 polymerase units of DNA Taq (Perkin Elmer)). using a plastic bar For the bacterial suspension, 4 μl of the cell suspension was added to 46 μl of the same PCR reaction mixture. For both strategies, the reaction mixture was covered with 50 μl of mineral oil and the PCR amplifications took using an initial denaturation step of 3 minutes at 95ac, followed by 30 cycles consisting of a denaturation step of 1 second at 95ac and a tempering step of 1 second at 55ac, in a thermal cycle apparatus JM Perkin Elmer 480 ™. The products of the amplification of PCR were then analyzed by standard agarose gel electrophoresis (2%). The amplification products were visualized on agarose gels containing 2.5 μg / ml of ethidium bromide under UV at 254 nm. The entire PCT trial can be completed in about an hour.

Alternatively, the amplification of the bacterial cultures was performed as described above, but using a "warm start" protocol. In this case, an initial reaction mixture containing the target DNA, the preparators and the dNTPs, was heated to 852C before the addition of the other components of the PCR reaction mixture. The final concentration of all the reagents was as described above. Next, the PCR reactions were presented to the thermal cycle and to the analysis, as described above.

EXAMPLE 8: Direct amplification of clinical specimens. For the amplification of urine specimens, 4 μl of undiluted or diluted urine (1:10) was added directly to 46 μl of the above PCR reaction mixture and amplified as described above. To improve bacterial cell lysis and eliminate the inhibitory effects of PCR on clinical specimens, samples were usually diluted in the lysis buffer containing detergent (s). Next, the lysate was added directly to the PCR reaction mixture. Heat treatments of the lysates, before DNA amplification, using a thermoclase or a microwave oven, can also be performed to increase the efficiency of cell lysis.

Our strategy is to develop fast and simple protocols to eliminate the inhibitory effects of PCR from clinical specimens and to lyse bacterial cells to perform DNA amplification directly from a variety of biological samples. PCR has the advantage of being compatible with crude preparations of DNA. For example, blood, cerebrospinal fluid and sera can be used directly in PCT assays after a brief heat treatment. We try to use such quick and simple strategies to develop rapid protocols for the amplification of DNA from a variety of clinical specimens.

EXAMPLE 9: Detection of antibiotic resistant genes The presence of antibiotic resistant genes, which are frequently found and clinically relevant, was identified using PCR or hybridization amplification protocols, described in previous sections. The specific oligonucleotides used as a basis for DNA-based tests are selected from the sequences of antibiotic-resistant genes. These tests can be performed either directly from clinical specimens or from a bacterial colony and should complement diagnostic tests for the specific identification of bacteria.

EXAMPLE 10 Same as in Examples 7 and 8, except that the assays were performed by multiplex PCR (ie using several pairs of preparators in a simple PCR reaction) to (i) reach a ubiquity of 100% for the specific target pathogen or (ii) simultaneously detect several species of bacterial pathogens. For example, detection of Escherichia coli requires three pairs of PCT preparers to ensure 100% ubiquity. Therefore, a multiplex PCR assay (using the "warm start" protocol (Example 7) with those three pairs of primers was developed.This strategy was also used for other bacterial pathogens for which more than one pair The preparation of multiplex PCR assays can also be used to (i) simultaneously detect several bacterial species or, alternatively, (ii) simultaneously identify bacterial pathogens and detect specific genes. resistant to antibiotics or directly from a clinical specimen or a bacterial colony For these applications, plicon detection methods must be adapted to differentiate the various amplicons produced The standard agarose gel electrophoresis can be used because it discriminates the ampli-cones based on their sizes.Another useful strategy for this purpose is the detection of A variety of fluorochromes emitted at different wavelengths each are coupled with a specific oligonucleotide linked to a fluorescence quencher, which is degraded during amplification to release the fluorochrome (eg TaqMan ™, Perkin Elmer).

EXAMPLE 11: Detection of Amplification Products. Those skilled in the art will appreciate that alternatives other than standard agarose gel electrophoresis (Example 7) can be used for the disclosure of the amplification products. Such methods may be based on detection of fluorescence after amplification (e.g. Amplisensor ™, Biotronics, TaqMan ™) or other labels, such as biotin (SHARP SIGNAL ™ system, Digene Diagnostics). These methods are quantitative and easily automatic. One of the amplification primers or an oligonucleotide internal probe specific to the amplicon (s) derived from fragment probes of specific species is coupled to the fluorochrome or to any other label. Methods based on the detection of fluorescence are particularly suitable for diagnostic tests, since they are fast and flexible as fluorochromes, which emit different wavelengths, are available (Perkin Elmer).

EXAMPLE 12 Amplification primers of antibiotic and universal resistant genes, of specific species, can be used in another rapid amplification procedure, such as the ligase chain reaction (LCR), transcription based amplification systems (TAS), self-sustained sequence reproductions (3SR), amplification based on nucleic acid sequence (NASBA), cord displacement amplification (SDA) and branched DNA (bDNA) or any other method to increase the sensitivity of the test. The amplifications can be performed from an isolated bacterial colony or directly from clinical specimens. The scope of this invention, therefore, is not limited to the use of PCR, but rather includes the use of any method to specifically identify bacterial DNA and which can be used to increase the speed and sensitivity of the tests.

EXAMPLE 13 A test kit will contain sets of specific probes "for each bacterium, as well as a set of universal probes.The equipment is provided in the form of test components, which consist of the set of universal probes labeled with non-radioactive labels, As well as specific probes labeled for the detection of each bacterium of interest in specific clinical samples, the equipment will also include the necessary test reagents to perform pre-hybridization, hybridization, washing steps and hybrid detection. for the detection of known antibiotic resistant genes (or derivatives thereof) will be included.Of course, the equipment will include standard samples that are to be used as negative and positive controls for each hybridization test. in the equipment will be adapted for each type of specimen and to detect pathogens commonly found in that kind of specimen. Reagents for universal bacteria detection will also be included. Based on the sites of infection, the following equipment for the specific detection of pathogens can be developed: • A team for the universal detection of bacterial pathogens of most clinical specimens, which contain sets of probes specific to highly conserved regions of bacterial genomes. «An equipment for the detection of bacterial pathogens, recovered from urine samples, containing eight specific test components (sets of probes for the detection of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Proteus mirabilis, Psudomonas aeruginosa, Staphylococcus saprophyticus, Staphylococcus aureus and Staphylococcus epidermidis). • A team for the detection of respiratory pathogens, which contains seven specific test components (sets of probes to detect Streptococcus pneumoniae. * Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus). • Equipment for the detection of pathogens recovered from blood samples, containing eleven specific test components (sets of probes for the detection of Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Staphylococcus epidermidis). «A team for the detection of pathogens that cause meningitis, which contains four specific test components (sets of probes for the detection of Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli and Pseudomonas aeruginosa).

• A team for the detection of antibiotic resistant genes, clinically important, which contains sets of probes for the specific detection of at least one of the following 19 genes, associated with bacterial resistance: klatem. klaro 't > lashv aadB, aacCl, aacC2, aacC3, aacA4, mecA, vanA, vanH, vanX, satA, aacA-aphD, vat, vga, msrA, sul and int. • Other equipment adapted for the detection of skin pathogens, abdominal wounds or other clinically relevant equipment that may develop.

EXAMPLE 14 Same as Example 13, except that the test equipment contains all the reagents and controls to perform DNA amplification assays. The diagnostic equipment will be adapted for amplification by the PCE (or other amplification methods), performed directly or from clinical specimens or a colony of bacteria. The components required for the universal detection of bacteria, the identification of bacteria and the detection of genes resistant to antibiotics will be included. The amplification assays can be performed in tubes or in microtiter plates, which have multiple wells. For plate assays, the wells will be coated with the specific amplification primers and the control DNAs and the detection of the amplification products will be automatic. The reagents and the preparation of the amplification pass the universal detection of bacteria will be included in the equipment for the tests carried out directly from the clinical specimens. the components required for bacterial identification and the detection of genes with resistance to antibiotics will be included in the equipment to be tested directly from the colonies, as well as in equipment to be tested directly from clinical specimens. The equipment will be adapted for use with each type of specimen, as described in Example 13, for diagnostic kits based on hybridization.

EXAMPLE 15 It will be understood that the use of the amplification probes and primers, described in this invention, for the detection and identification of bacteria, is not limited to clinical microbiology applications. In fact we believe that other sectors can also benefit from these new technologies. For example, these tests can be used by industries for the quality control of food, water, pharmaceutical products or other products that require microbiological control. These tests can also be applied to detect and identify bacteria in biological samples of organisms besides humans (for example, other primates, mammals, farm animals and livestock). These diagnostic tools can also be very useful for research purposes, which include clinical trials and epidemiological studies.

Table 1. Distribution of urinary isolates from positive urine samples (> 107 CFU / 1) at the "Center Hospitalier de l'Université Laval (CHUL)" for the period 1992-1994 % of Isolates Organisms November 92 April 93 July 93 January 94 n = 267a n = 265 n = 238 n = 281 Escherichia coli 53.2 51.7 53.8 54.1 Enterococcus faecalis 13.8 12.4 11.7 11.4 Klebsiella pneumoniae 6.4 6.4 5.5 5.3 Staphylococcus epidermidis 7.1 7.9 3.0 6.4 Proteus mirabilis 2.6 3.4 3.8 2.5 Pseudomonas aeruginosa 3.7 3.0 5.0 2.9 Staphylococcus saprophyticus 3.0 1.9 5.4 1.4 Othersb 10.2 13.3 11.8 16.0 an = total number of isolates for the indicated month b See Table 2 Table 2. Distribution of non-common urinary isolates from positive urine samples (> 107 CFU / 1) at the "Center Hospitalier de l'Université Laval (CHUL) "for the period 1992-1994 % of Isolates Organizations November 92 April 93 July 93 January 94 Staphylococcus aureus 0.4 1.1 1.3 1.4 Staphylococcus spp. 2.2 4.9 1.7 6.0 Micrococcus spp. 0.0. '0.0 0.4 0.7 Enterococcus faecium 0.4 0.4 1.3 1.4 Ci trobacter spp. 1..4 0.8 0.4 0.7 Enterobacter spp. 1.5 1.1 1.3 1.4 Klebsiella oxytoca. 1.1 1.5 2.5 1.8 Serrada spp. 0.8 0.0 0.5 0.0 Proteus spp. 0.4 0.4 0.0 1.1 Morganella and Providencia 0.4 0.8 0.4 0.0 Ha f nía alvei 0.8 0.0 0.0 0.0 NFBb 0.0 0.4 1.3 1.1 Candida spp. 0.8 1.9 0.7 0.4 a Non-common urinary isolates are those identified as Other "in Table 1. b NFB: non-fermentative bacilli (ie, Stenotrophomonas and Aclnetobacter Table 3. Distribution of positive urine specimens3 (bacterial count> 107CFU / 1) and negative (bacterial count <10 * 7 •. CFU / 1) tested at the "Center Hospitalier de l'Université Laval (CHUL)" for the period 1992-1994 • Number of isolates (%) Specimens November 92 April 93 July 93 January 94 Received: 1383 (100) 1338 (100) 1139 (100) 1345 (100) i positive: 267 (19.3) 265 (19.8) 238 (20.9) 281 (20.9) '1116 (80.7) 1073 (80.2) • 901 (79.1 ) 1064 (79.1) negative: a Based on standard diagnostic methods, the minimum number of bacterial pathogens in urine samples, to indicate a urinary tract infection, is usually 107 CFU / 1.

Table 4. Distribution of positive and negative clinical specimens tested in the CHUL Microbiology Laboratory.

No.% of% of Clinical Specimens to Specimens Specimens Urine 17,981 19.4 80.6 Blood culture / marrow 10,010 6.9 93.1 Saliva 1,266 68.4 31.6 10 Surface Pus 1,136 72.3 27.7 Cerebrospinal fluid 553 1.0 99.0 Synovial-joint fluid 523 2.7 97.3, Bronq, / Trachea / A íg. / Throat 502 56.6 43.4 Í Pus profunda 473 56.8 43.2 15 Ears 289 47.1 52.9 Pleural and pericardial fluid 132 1.0 99.0 Peritoneal fluid 101: 28.6 71.4 a Specimens tested from February 1994 to January 1995 • Table 5. Bacterial species (66) used to test the specificity of probes;, of DNA fragments, oligonucleotide probes and PCR primers No. of Bacterial Species strains Bacterial species tested strains tested Gram negative Gram negative Proteus mirabilis 5 Haemophilus for influenzae 2 Klebsiella pneumoniae 5 Bordetella pertussis 2 Pseudomonas aeruginosa 5 Haemophilus parahaemolyticus 2 Escherichia coli 5 Haemophilus haemolyticus 2 Moraxel la catarrhalis 5 Haemophilus aegyptius 1 Proteus vulgaris 2 Kingella indologenes 1 Mor tanella morganii 2 Moraxella atlantae 1 Enterobacter cloacae 2 Neisseria caviae 1 Providence stuartii 1 Neisseria subflava 1 Providence species 1 Moraxella urethralis 1 Enterobacter agglo erans 2 Shigella sonnei 1 Providencia rettgeri 2 Shigella flexneri 1 Neisseria mucosa 1 Klebsiella oxytoca 2 Providencia alcalifaciens 1 Serratia marcescens 2 Providencia rustigianii 1 Salmonella typhimurium 1 Burkholderia cepacia 2 Yersinia enterocolitica 1 Enterobacter aerogenes 2 Acinetobacter calcoaceticus 1 Stenotrophomonas maltophilia 2 Acinetobacter lwoffi 1 Pseudomonas fluorescens 1 Hafnia alvei 2 Co ammonium acidovorans 2 Citrobacter diversus 1 Pseudomonas putida 2 Citrobacter freundii 1 Haemophi lus influenzae 5 Salmonella species 1 Continue on the next page Table 5. (Continued) Bacterial species (66) used to test the specificity of probes from DNA fragments, oligonucleotide probes and PCR primers • No. of Bacterial Species strains tested Gram positive Streptococcus pneumoniae 7 Streptococcus salivarius 2 Streptococcus viridans 2 Streptococcus pyogenes 2 Staphylococcus aureus. 2 Staphylococcus epidermidis 2"Staphylococcus saprophyticus 5 Micrococcus species 2 Corynebacterium species 2 Streptococcus groupe B 2 Staphylococcus simulans 2 Staphylococcus ludgunensis 2 Staphylococcus capitis 2 Staphylococcus haemolyticus 2 Staphylococcus hominis 2 Enterococcus faecalis 2 Enterococcus faecium 1 Staphylococcus warneri 1 Enterococcus durans 1 Streptococcus bovis 1 Diphteroids 1 Lactobacillus ac'idophilus 1 Table 6. Species - specific DNA fragments and oligonucleotide probes for hybridization Organisms3 Number of fragment probes13 Number of oligonucleotide probes Tested Ubiquitous Synthesized Ubiquitous Specifics E. coli d - 20 12 E. coli * 14 K. pneumoniae® - 15 1 1 K. pneumoniae 33 18 12 8 P. mirabilis®- - 3 3 2 P. mirabilis 14 15 8 7 P. aeruginosa®- - 26 13 9 P. aeruginosa 6 2 2e 6 0 0 S. saprophyticus 4 4 20 H. influenzae® 16 2 2 H. influenzae 20 1 1 S. pneumoniae®- 6 1 1 S. pneumoniae 19 2 2 4 1 1 M. catarrhalis 2 2 2 S. epidermidis 62 1 1 S. aureus 30 1 1 Universal probes "79 a No probe of DNA fragments or oligonucleotides was tested for E. faecalis and S-pyogenes. . b The sizes of the DNA fragments vary from 0.25 to 5.0 kbp. c A specific probe was considered ubiquitous when at least 80% of the isolates of the target species (approximately 80 isolates) were recognized for each specific probe. When 2 or more probes are combined, 100% of the isolates are recognized. d These sequences were selected from the data banks. • e Ubiquity tested with approximately 10 isolates of target species f A majority of probes (8/9) do not discriminate E coli and Shigella spp. g Evidence of ubiquity with a set of 7 probes detected all 66 bacterial species listed in Table 5.

• Table 7. PCR amplification for bacterial pathogens commonly found in urine, saliva, blood, cerebrospinal fluid and other specimens.

• Size of Amplification of Organism Preparing partner3 Amplicon (bp) Ubiquity13 DNA of # (SEQ ID NO) Speciescolony0 Menes0 't and. coli 1 * (55 + 56) 107 75/80 + + 2e (46 + 47) 297 77/80 + + 3 (42 + 43) 102 78/80 + + 4 (131 + 132) 134 73/80 + + 1 + 3 + 4 - 80/80 + + E. faecalis le (38 + 39) 200 71/80 + + 2ß (40 + 41) 121 79/80 + + 1 + 2 - 80/80 + + K. pneumoniae 1 (67 + 68) 198 76/80 + + 2 (61 + 62) 143 67/80 + + 3 »(135 + 136) 148 78/80 + N.T. 4 (137 + 138) 116 69/80 + N.T. 1 + 2 + 3 - 80/80 + N.T. P. mirabilis 1 (74 + 75) 167 73/80 + N.T. 2 (133 + 134) 123 80/80 + N.T. P. aeruginosa le (83 + 84) 139 79/80 + N.T. 2ß (85 + 86) 223 80/80 + N.T.

S. saprophyticus 1 (98 + 99) 126 79/80 + + 2 (139 + 140) 190 80/80 + NT H. catarrhalis 1 (112 + 113) 157 79/80 + NT 2 (118 + 119) 118 80/80 + NT 3 (160 + 119) 137 80/80 + NT • H. influenzae le (154 + 155) 217 80/80 + NT S. pneumoniae le (156 + 157) 134 80/80 + NT 2β (158 + 159) 197 74/80 + NT 3 (78 + 79) 175 67/80 + NT twenty continued on the next page Table 7 (Continued). PCR amplification for bacterial pathogens commonly found in urine, saliva, blood, cerebrospinal fluid and other specimens.

Size of Organism Amplification Preparer Couple3 Amplicon (bp) Ubiquity DNA # (SEQ ID NO) Species- colonlas0 menesd S. epidermidis 1 (147 + 148) 175 80/80 N. .T. 2 (145 + 146) 125 80/80 N. .T.

S. aureus 1 (152 + 153) 108 80/80 N. .T. 2 (149 + 150) 151 80/80 N, .T. 3 (149 + 151) 176 80/80 N., t.

S. pyogenes - le (141 + 142) 213 80/80 N, .T. 2e (143 + 144) 1S7 24/24 N. .T.

Universal (126-127) 241 194/195 ^ a All of the preparator pairs are specific in the PCR assays, since amplification was not observed with the DNA of 66 different species of both Gram positive and Gram negative bacteria in addition to the species of interest (Table 5). b Ubiquity was normally tested on 80 strains of the species of interest. All retained breeding pairs amplified at least 90% of isolates. When combinations of the preparers are used, a ubiquity of 100% was reached. c For all breeding pairs and multiple combinations, PCR amplifications performed directly from a bacterial colony were 100% specific species. d PCR assays were performed directly from urine specimens. e The prepared couples derived from the sequences of data banks. These groomers without "e" are derived from our fragments of specific species. f For S. pyogenes, partner # 1 is specific for Group A Streptococci (GAS).

Preparation partner # 2 is specific to the exotoxin A gene that produces GAS (SpeA). 9 Ubiquity tested in 195 isolates from 23 species representative of bacterial pathogens commonly found in clinical specimens. n Optimizations are in progress to eliminate the non-specific amplification observed with some bacterial species in addition to the target species. N.T .: Not tested.

Table 8. Selected genes of antibiotic resistance for diagnostic purposes Antibiotic Genes Bacteria3 SEQ ID NO. \ lblatem) TEM-1 ß- lac tajos Sp terobac ter iaceae. 161 1 Pseudomonadaceae. Haemophilus, Neisseria ibblarob) ROB-1 ß-lactams Haemophilus, Pasteurella 162 (blashv) SHV-1 ß-lactams Klebsiella and other 163 • Enterobacteriaceae 1 aadB. aacCl, aac 2. Aminoglycos ides Enterobacteriaceae, 164, 165. 166 aacC2, aacA4 Pseudomonadaceae 167. 168 jpecA ß-lactams Staphyl ococci 169 vanH. veunA, VanX Vancoraycin Enterococci 170 sa tA Macrolides Enterococci 173 aa A-aphD Aminoglycosides Enteroc cci, 174 Staphylococci v Macro ides Staphyl ococci 175 vga Macro lides Staphyl ococci 176 msrA Eryt romycin Staphylococci 177 sequences ß-lactams, trimethoprim, 1 Enterobacteriaceae, -171 . 172 preserved aminoglycosides, anti Pseudomonadaceae Int and septic Sul, chloroanphenicol 1 Bacteria that have a high incidence for specific genes of antibiotic resistance. The presence of other bacteria is not excluded Annex I: oligonucleotide probes, specific and ubiquitous, for hybridization SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Nucleotide Position Bacterial Species: Escherichia coli 44 5 '-CAC CCG CTT GCG TGG CAÁ GCT GCC C 5a 213-237 45 5 '-CGT TTG TGG ATT CCA GTT CCA TCC G 5a 489-513 48 5 '-TGA AGC ACT GGC CGA AAT GCT GCG T 6a 759-783 0 49 5' -GAT GTA CAG GAT TCG TTG AAG GCT T 6a 898-922 50 5'-TAG CGA AGG CGT AGC AGA AAC TAA C 7a 1264-1288 51 5 '-GCA ACC CGA ACT CA CA CGC CGG ATT T 7a 1227-1251 52 5 '-ATA CAC AAG GGT CGC ATC TGC GGC c 7a 1313-1337 53 5 '-TGC GTA TGC ATT GCA GAC CTT GTG GC 7a 111-136 54 5"-GCT TTC ACT GGA TAT CGC GCT TGG G 7a 373-397 Bacterial species:, Proteus mirabilis 70b 5 '-TGG TTC ACT GAC TTT GCG ATG TTT C 12 23-47 71 5 '-TCG AGG ATG GCA TGC ACT AGA AAA T 12 53-77 72b 5 '-CGC TGA TTA GGT TTC GCT AAA ATC TTA TTA 12 80-109 73 5 '-TTG ATC CTC ATT TTA TTA ATC ACÁ TGA CCA 12 174-203 a Databank sequences ^ These sequences are from the opposite strand of DNA from the sequences given in the Sequence list Annex X: Oligonucleotide probes, specific and ubiquitous, for hybridization SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Position of Nucieotide Bacterial species:. Proteus mirabilis 76 5 '-CCG CCT TTA GCA TTA ATT GGT GTT TAT AGT 13 • 246-275 77 -. 77 - 5 * -CCT ATT GCA GAT ACC TTA AAT GTC TTG GGC 13 291-320 Sfj 5 '-TTG AGT GAT GAT TTC ACT GAC TCC C 14 18-42 81 5' -GTC AGA CAG TGA TGC TGA CGA CAC A 15a 1185-1209 82 5 '-TGG TTG TCA TGC TGT TTG TGT GAA AAT 15a 1224-1250 Bacterial species: • 'Klebsiella pneumoniae 57 5' -GTG GTG TCG TTC AGC GCT TTC AC 8 45-67 58 5 '-GCG ATA TTC ACA CCC TAC GCA GCC A • 9 161-185 59 .5' -GTC GAA AAT GCC GGA AGA GGT ATA CG 9 203-228 6O13 5"-ACT GAG CTG CAG ACC GGT AAA ACT CA 9 233-258! 63 ^ 5 '-CGT GAT GGA TAT TCT TAA CGA AGG GC 10 250-275 64b 5' -ACC AAA CTG TTG AGC CGC CTG GA 10 201-223 65 5 '-GTG ATC GCC CCT CAT CTG CTA CT 10 77-99 66 5 * -CGC CCT TCG TTA AGA ATA TCC ATC AC 10 249-274 69 5' -CAG GAA GAT GCT GCA CCG GTT GTT G 11a 296-320 Databank sequences ^ These sequences are from the opposite strand of DNA from the sequences given in the Sequence list Annex X: Oligonucleotide probes, specific and ubiquitous, for hybridization • SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Nucleotide Position Bacterial Species: (Pseudomonas aeruginosa 87 5 '-AAT GCG GCT GTA CCT CGG CGC TGG T 18a 2985-3009 88 5 '-GGC GGA GGG CCA GTT GCA CCT. GCC A 18a 2929-2953 • 89 5 '-AGC CCT GCT CCT CGG CAG CCT CTG C 18a 2821-2845 90 5 '-TGG CTT TTG CAÁ CCG CGT TCA GGT T 18a 1079-1103 91 5 '-GCG CCC GCG AGG GCA TGC TTC GAT G 19a 705-729 92 5 * -ACC TGG GCG CCA ACT ACA AGT CT A 19a 668-692 93 5' -GGC TAC GCT GCC GGG CTG CAG GCC G 19a 505- 529 94 5 '-CCG ATC TAC ACC ATC GAG ATG GGC G 20a 1211-1235 95 5' -GAG CGC GGC TAT GTG TTC GTC GGC T 20a 2111-2135 Bacterial species: < Streptococcus pneumoniae 120 5 '-TCT GTG CTA GAG ACT GCC CCA TTT C 30 423-447 121 5' -CGA TGT CTT GAT TGA GCA GGG TTA T 31a 1198-1222 Databank sequences b These sequences are from the opposite strand of DNA of the sequences given in the Sequence list Annex X: Oligonucleotide probes, specific and ubiquitous, for hybridization SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Nucleotide Position Bacterial Species: Staphylococcus p "» rophyticus 96 5 * -CGT TTT TAC CCT TAC CTT TTC GTA CTA CC 45-73 97b 5 '-TCA GGC AGA GGT AGT ACG AAA AGG TAA GGG 21 53-82 100 5 * -CAC CAÁ GTT TGA CAC GTG AAG ATT CAT 22 89-115 101 * 3 5 '-ATG AGA GAA GCG GAG TCA GAT TAT GTG CAG 23 105-134 102 5 '-CGC TCA TTA CGT ACÁ GTG ACÁ ATC G 24 20-44 103 5 '-CTG GTT AGC TTG ACT CTT AAC AAT CTT GTC 24 61-90 10 5"-GAC GCG ATT GTC ACT GTA CGT AAT GAG CGA 24 19-48 Bacterial Species: Moraxella catarrhalis I 108 5 '-GCC CCA AAA CAA TGA AAC ATA TGG T 28 81-105 109 5 '-CTG CAG ATT TTG GAA TCA TAT CGC C 28 126-150 110 5 '-TGG TTT GAC CAG TAT TTA ACG CCA T 28 165-189 111 5 '-CAÁ CGG CAC CTG ATG TAC CTT GTA C 28 232-256 114 5 * -TTA CAÁ CCT GCA CCA CAÁ GTC ATC A 29 97-121 115 5 '-GTA CAÁ ACÁ AGC CGT CAG CGA CTT A 29 139-163 116 5 '-CAÁ TCT GCG TGT GTG CGT TCA CT 29 178-200 117 5 '-GCT ACT TTG TCA GCT TTA GCC ATT CA 29 287-312 Databank sequences bThese sequences are from the opposite strand of DNA from the sequences given in the Sequence list Annex X: Oligonucleotide probes, specific and ubiquitous, for hybridization SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Nucleotide Position Bacterial Species: Haemophilus influenzae 105 ^ 5 '-GCG TCA GAA AAA GTA GGC GAA ATG AAA G 25 138-165 106 5- -AGC GGC TCT ATC TTG TAA TGA CAC A 26a 770-794 107b 5' -GAA ACG TGA ACT CCC CTC TAT ATA A 27a 5184-5208 Universal Probes0 (0 122b 5"-ATC CCA CCT TAG GCG GCT GGC TCC A 123 5" -ACG TCA AGT CAT CAT GGC CCT TAC GAG TAG G 12 5 '-GTG TGA CGG GCG GTG TGT ACÁ AGG C 125b 5' -GAG TTG CAG ACT CCA ATC CGG ACT ACG A 128 ^ 5 '-CCC TAT ACÁ TCA CCT TGC GGT TTA GCA AG-129 5' -GGG GGG ACC ATC CTC CAA GGC TAA ATA C 5 13? B 5 '-CGT CCA CTT TCG TGT TTG CAG AGT GCT GTG TT - a Databank sequences b These sequences are from the opposite strand of DNA of the 0 sequences given in the Sequences list c Universal probes are derived from 16S or 23S ribosomal RNA gene sequences, not included in the list of Sequences Annex II : oligonucleotide probes, specific and ubiquitous, for amplification SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Nucleotide Position • Bacterial Species: Escherichia coli 42 5 '-GCT TTC CAG CGT CAT ATT G 4 177-195 • 43b 5 '-GAT CTC GAC AAA ATG GTG A 4 260-278 46 5 '-TCA CCC GCT TGC GTG GC 5a 212-228 47b 5' -GGA ACT GGA ATC CAC AAA C 5a 490-508 55 5 '-GCA ACC CGA ACT CAA CGC C 7a 1227-1245 56b 5 '-GCA GAT GCG ACC CTT GTG T 7a 1315-1333 131 5 '-CAG GAG TAC GGT GAT TTT TA 3 60-79 132b 5 '-ATT TCT GGT TTG GTC ATA CA 3 174-193 Bacterial Species: Enterococcus faecalis 38 5 '-GCA ATA CAG GGA AAA ATG TC Ia 69-88 39b 5 '-CTT CAT CAÁ ACÁ ATT AAC TC Ia 249-268 40 5 ', - GAA CAG AAG AAG CCA AAA AA 2a 569-588 41b 5 '-GCA ATC CCA AAT AAT ACG GT 2a 670-689 a Databank sequences bThese sequences are from the opposite strand of DNA from the sequences given in the Sequence list Annex II: Oligonucleotide probes, specific and ubiquitous, for amplification SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Nucleotide Position Bacterial Species: Klebsiella pneumoniae 61 5 '-GAC AGT CAG TTC GTC AGC C 9 37-55 62b 5 '-CGT AGG GTG TGA ATA-TCG C 9 161-179 67 5 '-TCG CCC CTC ATC TGC TAC T 10 81-99 68b 5 '-GAT CGT GAT GGA TAT TCT T 10 260-278 135 5'-GCA GCG TGG TGT CGT TCA 8 40-57 136b 5 '-AGC TGG CAÁ CGG CTG GTC 8 170-187 137 5 '-ATT CAC ACC CTA CGC AGC CA 9 166-185 138b 5 '-ATC CGG CAG CAT CTC TTT GT 9 262-281 Bacterial Species: i proteus mirabilis 74 5 '-GAA ACÁ TCG CAÁ AGT CAG T 12 23-41 75 5 '-ATA AAA TGA GGA TCA AGT TC 12 170-189 133 5 '-CGG GAG TCA GTG AAA TCA TC 14 17-36 134b 5 '-CTA AAA TCG CCA CAC CTC TT 14 120-139 a Databank sequences bThese sequences are from the opposite strand of DNA from the sequences given in the Sequence list Annex XX: Oligonucleotide probes, specific and ubiquitous, for amplification SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Nucleotide Position Bacterial Mirror: Staphylococcus saprophyticus 98 5"-CGT TTT TAC CCT TAC CTT TTC GTA CT 21 45-70 99b 5 '-ATC GAT CAT CAC ATT CCA TTT GTT TTT A 21 143-170 GTT AGC TTG ACT CTT AAC AAT C 24 61-85 14? B 5' -TCT TAA CGA TAG AAT GGA GCA ACT G 24 226-250 Bacterial Species: Pseudomonas aeruginosa 83 5 '-CGA GCG GGT GGT GTT CAT C 16a 554-572 84b 5 '-CAÁ GTC GTC GTC GGA GGG A 16a 674-692 85 5 '-TCG CTG TTC ATC AAG ACC C 17a 1423-1441 86b 5 '-CCG AGA ACC AGA CTT CAT C 17a 1627-1645 Bacterial species: Moraxella catarrhalis 112 5 '-GGC ACC TGA TGT ACC TTG 28 235-252 113b 5' -AAC AGC TCA CAC GCA TT 28 375-391 118 5 '-TGT TTT GAG CTT TTT ATT TTT TGA 29 41-64 119b 5' -CGC TGA CGG CTT GTT TGT ACC A 29 137-158 160 5 '-GCT CAA ATC AGG GTC AGC 29 22-39 119b 5"-CGC TGA CGG CTT GTT TGT ACC A 29 137-158 20 a Databank sequences bThese sequences are from the opposite strand of DNA of the sequences given in the list of Sequences Annex II: Oligonucleotide probes, specific and ubiquitous, for amplification SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Position of Nucieotide Bacterial Species: Staphylococcus epidermidis 145 5 '-ATC AAA AAG TTG GCG AAC CTT TTC A 36 21-45 146b 5 --CA AAG AGC GTG GAG AAA AGT ATC A 36 121-145 147 5 '-TCT CTT TTA ATT TCA TCT TCA ATT CCA TAG_36_448-477 148b 5 > -AAA CAC AAT TAC AGT CTG GTT ATC CAT ATC 36 593-622 Bacterial Species: Staphylococcus aureus 14g 5 '-CTT CAT TTT ACG GTG ACT TCT TAG AAG ATT 37 409-438 150 5 '-TCA ACT GTA GCT TCT TTA TCC ATA CGT TGA 37 288-317. 149b 5 > _CTT CAT TTT ACG GTG ACT TCT TAG AAG ATT 37 409-438 151 5 '-ATA TTT TAG CTT TTC AGT TTC TAT ATC AAC 37 263-292 152 5'-AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG 37 5-34 153 5 '-CGT AAT GAG ATT TCA GTA GAT AAT ACÁ ACÁ 37 83-112 a Databank sequences bThese sequences are from the opposite strand of DNA from the sequences given in the Sequence list Annex II: Oligonucleotide probes, specific and ubiquitous, for amplification SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Nucleotide Position Bacterial species: Haemophilus influenzae i 154 5 '-TTT AAC GAT CCT TTT ACT CCT TTT G 27a 5074-5098 155 5' -ACT GCT GTT GTA AAG AGG TTA AAA T 27a 5266-5290 Bacterial Species: Streptococcus pneumoniae 78 5 '-AGT AAA ATG AAA TAA GAA CAG GAC AG 34 164-189 79 5 '-AAA ACÁ GGA TAG GAG AAC GGG AAA A 34 314-338 156 5 '-ATT TGG TGA CGG GTG ACT TT 31a 1401-1420 157b 5 '-GCT GAG GAT TTG TTC TTC TT 31a 1515-1534 158 5 '-GAG CGG TTT CTA TGA TTG TA 35a 1342-1361 159b 5 * -ATC TTT CCT TTC TTG TTC TT 35a 1519-1538 E Cspecie Bacteriana: 1 Streptococcus pyogenes 141 5 * -TGA AAA TTC TTG TAA CAG GC 32a 286-305 142 5 '-GGC CAC CAG CTT GCC CAÁ TA 32a 479-498 143 5 '-ATA TTT TCT TTA TGA GGG TG 33a 966-985 1 b 5 '-ATC CTT AAA TAA AGT TGC CA 33a 1103-1122 a Databank sequences bThese sequences are from the opposite strand of DNA from the sequences given in the Sequence list Annex XX: oligonucleotide probes, specific and ubiquitous, for amplification SEQ ID NO. Nucleotide Sequence DNA fragment originating SEQ ID NO Nucleotide Position Universal Preparators0 '... i 126 5' -GGA GGA AGG TGG GGA TGA CG 127b 5 '-ATG GTG TGA CGG GCG GTG TG a Databank sequences bThese sequences are from the opposite strand of DNA from the sequences given in the Sequence list c The Universal primers that were derived from the sequence of the 16S ribosomal RNA gene are not included in the Sequence list.

Annex III. selection of universal probes by alignment of bacterial ribosomal RNA gene sequences 16S and 23S Reverse cord of SEQ ID. No: 122 TGGAGCC? AccscCTAA j GGTGGGAT 1461 1510 Streptococcus salivarius TGAGGTAACC TTTTGßAGCC AGCCGCCTAA GGTGGGATAG ATGANNGGGG .Proteus vulgaris TAGCTTAACC TTCGGOAGGG CGCTTACCAC TTTGTGATTC ATGACTGGGG Pseudomonas aeruginosa TAGTCTAACC GCAAGGOGGA CGGTTACCAC GGAGTGATTC ATGACTGGGG Neisseria gonorrhoeae TAGGGTAACC GCAA00AOTC CGCTTACCAC SSTA GC TC ATGACTGGGG Streptococcus lactis TTGCCTAACC GCAAGGAGGG CGCTTCCTAA GGTAAGACCG ATGACNNGGG Annex III. Selection of universal probes by alignment of bacterial ribosomal RNA gene sequences 16S and 23S SEQ ID. No: 123 ACGTCAAGTC? TC? TGGC CCTTACGAGT AGG 1251 1300 Haemophilus influenzae GGTNGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGAGT AGGGCTACAC Neiseeria gonorrhoeae GGTGGGGATG ACGTCAAGTC .. CTCATGGC CCTTATGACC AGGGCTTCAC Pseudomonas cepacia GGTNGGGATG ACGTCAAGTC .. CTCATGGC CCTTATGGGT AGGGCTTCAC Serratia marcescens GGTGGGGATG ACGTCAAOTC ..ATCATQGC Escherichia coli CCTTACQAGT AGGGCTACAC GGTGGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGACC AGGGCTACAC Proteus vulgaris GGTGGGGATG ACGTTAAGTC GTATCATGGC CCTTACGAGT AGGGCTACAC Pseudomonas aeruginosa GGTGGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGGCN AGGGCTACAC Clostridium perfringens GGTGGGGATG ACGTNNAATC ..ATCATGCC CNTTATGTGT AGGGCTACAC Mycoplasma hominis GGTGGGGATG ACGTCAAATC ..ATCATQCC TCTTACGAGT GGGGCCACAC Helicobacter pylori GGTGGGGACG ACGTCAAGTC ..ATCATGGC CCTTACGCCT AGGGCTACAC Mycoplasma pneumoniae GGAAGGGATG ACGTCAAATC ..ATCATGCC CCTTATGTCT AGGGCTGCAA 70 • Annex III. Selection of universal probes by alignment of bacterial ribosomal ARK gene sequences 16S and 23S Inverse of SEQ ID. No: 124 GCCTTGTACA CACCGCCCGT CACAC 1451 1490 Escherichia coli ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG Neisseria gonorrhoeae ACGTTCCCNG NNCTTOTACA CACCGCCCOT CACACCATGG Pseudomonas cepacia ACGTTCCCGG GTCTTOTACA CACNGCCCGT CACACCATGG Serratia marcescens ACGTTCCCGG GCCTTGTACA CACCQCCCGT CACACCATGG Proteus vulgaris ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG Haemophilus influenzae ACGTTCCCGG GCNTTGTACA CACCGCCCGT CACACCATGG Pseudomonas aeruginosa ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG Clostridium perfringens ACGTTCCCNG GTCTTGTACA CACCGCNCGT CACACCATGA Mycoplasma hominis ACGTTCTCGG GTCTTGTACA CACCGCCCGT CACACCATGG Helicobacter pylori ACGTTCCCGG GTCTTGTACT CACCGCCCGT CACACCATGG Mycoplasma pneumoniae ACGTTCTCGG GTCTTGTACA CACCGCCCGT CAAACTATGA Annex III. selection of universal probes by alignment of bacterial ribosomal RNA gene sequences 16S and 23S Reverse cord of SEQ ID. No: 125 TCG TAGTCCGGAT TGGAGTCTGC AACTC 1361 1400 Escherichia coli AAGTGCGTCG TAGTCCQQAT Tas? GTCTac AACTCGACTC Neisseria gonorrhoeae AAACCGATCG TAGTCCGGAT TGCACTCTGC AACTCGAGTG Pseudomonas cepacia AAACCGATCG TAGTCCGGAT TGCACTCTGC AACTCGAGTG Serratia marcescens AAGTATGTCG TAGTCCOGAT TGGAGTCTGC AACTCGACTC Proteus vulgaris AAGTCTGTCO TAGTCCGGAT TGQAsTCTGC AACTCGACTC Haemophilus influenzae AAGTACGTCT AAGTCCGGAT TGGAGTCTGC AACTCGACTC Pseudomonas aeruginosa AAACCGATCG TAGTCCOOAT CsCAGTCTGC AACTCGACTG Clostridium perfringens AAACCAGTCT CAOTTCOGAT TGTAOGCTGA AACTCGCCTA Mycoplasma ho inis AAGCCGATCT CAGTTCOOAT TGGAGTCTsC AATTCGACTC Helicobacter pylori ACACC ..TCT CAGTTCGGAT TGTAsacTGc AACTCGCCTG Mycoplasma pneumoniae AAGTTGGTCT CAGTTCGGAT TGAGGGCTGC AATTCGTCCT 72 ^ Annex III. Selection of universal probes by alignment of gene sequences of bacterial ribosomal RNA 16S and 23S reverse cord of SEQ ID. No: 128 CT CTCTGCTAAA CCGCAAGGTG ATGTATAGGG 1991 2040 Lactobacillus lactis AAACACAGCT CTCTGCTAAA CCGCAAGGTG Escherichia coli ATGTATAGGG GGTGACGCCT AAACACAGCA CTGTGCAAAC ACGAAAQTQG ACGTATACGG TGTGACGCCT AAACACAGCA CTCTGCAAAC Pseudomonas aeruginosa Pseudomonas cepacia ACGAAAQTGG ACGTATAGGG TGTGACGCCT AAACACAGCA CTCTGCAAAC ACsAAAsTOQ ACGTATAGGG TGTGACGCCT Bacillus stearother ophilus AAACACAGGT CTCTGCGAAG TCOTAAGOCß ACGTATAGGG GCTGACACCT Micrococcus luteus AAACACAGGT CCATGCGAAG TCOTAAGACG ATGTATATGQ ACTGACTCCT SEQ ID NO: 129 ssssGsAcc ATCCTCCAAG GCTAAATAC 481 530 Escherichia coli TGTCTGAATA TGGGGGGACC ATCCTCCAAQ QCTAAATACT i CCTGACTGAC TGTCTGAACA TGGGGGGACC Pseudomonas aeruginosa Pseudomonas cepacia ATCCTCCAAG GCTAAATACT ACTGACTGAC TGTCTGAAGA TGGGGGGACC ATCCTCCAAG GCTAAATACT CGTGATCGAC Lactobacillus lactis AGTTTGAATC CGGGAGGACC ATCTCCCAAC CCTAAATACT CCTTAGTGAC Micrococcus luteus CGTGTGAATC TGCCAGGACC ACCTGGTAAG CCTGAATACT ACCTGTTGAC 73 • Annex III. Selection of universal probes by alignment of bacterial ribosomal RNA gene sequences 16S and 23S Reverse cord of SEQ ID. Ko: 130 AACACAGCA CTCTGCAAAC ACGAAAGTGG ACG 1981 2030 Pseudomonas aeruginosa TGTTTATTAA AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG Escherichia coli TGTTTATTAA AAACACAGCA CTGTQCAAAC ACGAAAGTGG ACGTATACGG Pseudomonas cepacia 'TGTTTAATAA AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG Bacillus stearother ophilus TGTTTATCAA AAACACAGGT CTCTGCGAAG TCGTAAGGCG ACGTATAGGG Lactobacillus lactis TGTTTATCAA AAACACAGCT CTCTGCTAAA CCGCAAGGTG ATGTATAGGG Micrococcus luteus TGTTTATCAA AAACACAQGT CCATGCGAAG TCGTAAGACG ATGTATATGG 74"• • Annex IV. Selection of universal probes by alignment of bacterial ribosomal RNA gene sequences 16S and 23S SEQ ID No. 126 GGAGGAA GGTGGGGATG ACG 5 Inverse cord of SEQ ID. No: 127 GA CACCGCCCGT CACACCAT 1241 1270 1461 1490 Escherichia coli ACTGOAGGAA ßOTOOGOATG ACOTCAAGTC GCCTTGTACA CACCGCCCOT CACACCATGG Neisseria gonorxhoeae Gccos? Ss ?? QQ OasOATG ACOTCAAGTC NNCTTGTAC? CACCOCCCOT CACACCATGG Pseudomonas cepacia ACCGOAOGAA GOTNOGOATG ACOTCAAGTC ...... GTCTTGTAC? C? CNGCCCGT C? C? CC? TGG 10 Serra tia marcescens Ac GO? Sß ?? OOTOOGOATO ACOTCAAGTC GCCTTGTACA C? CCOCCCOT CACACC? TGG Proteus vulgaris ACCGO? GO ?? ootsass? to ACOTTAAGTC GCCTTGTACA CACCOCCCOT CACACCATGG Haemophilus influenzae ACTGO? GG ?? GOTNOOGATO ACOTCAAGTC GCNTTGTAC? CACCOCCCOT CACACCATGG Legionella pneumophila ACCGsAGGA? GGCGsso? Ts? CGTCAAGTC GCCTTGTACA CACCGCCCOT CAC? CCATGG Pseudomonas aeruginosa ACCGO? GG ?? OaTOGOGATG ACGTCAAGTC GCCTTGTAC? CACCGCCCOT CACACCATGG Clostridium perfringens CCAGGAGGAA GOTOOGOATG ACGTNNAATC GTCTTGTACA CACCGCNCGT CACACCATGA Mycoplasma hominis CTGGGAGGAA GGTGGGGATG ACOTCAAATC GTCTTGTAC? CACCGCCCOT CACACC? TGG Helicobacter pylori GGAGGAGG ?? sOTGGGG? CG ACOTCAAGTC GTCTTGTACT CACCGCCCGT CACACC? TGG Mycoplasma pneumoniae ATTGGAGGAA GGAAGGG? Ts? CGTCAAATC GTCTTGTACA CACCGCCCGT CAAACTATGA 75 •

Claims (29)

1. CLAIMS • 1. A method for the detection of target bacterial species, which is suspected to be present in a sample, characterized in that: a) The nucleic acids of the target bacterial species are contacted with at least one pair of amplification primers , i) the pair of preparers is derived from a • 10 fragment of bacterial DNA that is specific for given target bacterial species and that is ubiquitous for the chains of the target bacterial species given, ii) when required, the pair of 15 preparers of i) are grouped with another pair of preparers, to achieve a ubiquity of 100%, the other pair of primers is derived from another bacterial DNA fragment that is specific and ubiquitous for the target bacterial species given, lii) the pairs of amplification primers of i) and ii) are specific and ubiquitous for the given target bacterial species. iv) all the amplification primers are selected to have a given amplification annealing temperature, for hybridization with the nucleic acids of target bacterial species, iflf the given amplification annealing temperature allows the multiple amplification to be performed; 5 b) allow the amplification to proceed under given amplification conditions including the annealing temperature, and c) detect the presence or amount of a given amplified sequence as an indication of the • 10 presence or quantity of given bacterial species.
2. 2. A method as defined in claim 1, further comprising the use of universal amplification primers to determine the presence or amount of nucleic acids of any bacterial species, 15 where universal bacterial detection is performed under the same amplification conditions. 3. A method as defined in claim 1 or 2, further comprising the use of amplification primers to determine the presence or amount of 20 nucleic acids of an antibiotic-resistant gene, wherein the detection of an antibiotic-resistant gene is carried out under the same amplification conditions. 4. A method as defined in any of claims 1 to 3, wherein the bacterial species is 25 selected from Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae, Moraxella catarrhalis, the gene of exotoxin A that produces group A Streptococcus SpeA and any of its combinations. 5. A method as defined in claim 3 or 4, wherein the antibiotic-resistant gene is selected from blatem, blar? B / blashb / aadB, aacCl, aacC2, aacC3, aacA4, mecA, va A, vanH, vanX , satA, aacA-aphD, vat, vga, msrA, sul, Int and any of their combinations. 6. A method as defined in any of claims 1 to 5, wherein the bacterial DNA fragment is selected from: SEQ ID NO.
3. 3, SEQ ID NO.
4. 4, SEQ ID NO.
5. 5, SEQ ID NO.
6. 6, SEQ ID NO. 7 and a complementary sequence thereof, to determine the presence or amount of Escherichia coli; SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID DO NOT. 11, and a complementary sequence thereof to determine the presence or amount of Klebsiella pneumoniae; SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID 19, SEQ ID NO. 20 and a complementary sequence thereof, to determine the presence or amount of Pseudomonas aeruginosa; SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 and a complementary sequence thereof, to determine the presence or amount of Proteus mirabillis; SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 35 and a complementary sequence thereof, to determine the presence or amount of Streptococcus pneumoniae; SEQ ID NO. 37 and a complementary sequence thereof, to determine the presence or amount of Staphyl ococcus aureus; SEQ ID NO. 36 and a complementary sequence thereof, to determine the presence or amount of Staphylococcus epidermidis; SEQ ID NO. 1, SEQ ID NO. 2 and a complementary sequence thereof, to determine the presence or quantity of Enterococcus faecalis; SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, and a complementary sequence thereof, to determine the presence or amount of Staphylococcus saprophyticus; SEQ ID NO. 33, and a complementary sequence thereof, to determine the presence or quantity of bacterial species that carry the speA exotoxin A gene; SEQ ID NO. 32 and a complementary sequence thereof, to determine the presence or amount of • Streptococcus pyogenes; SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID No. 27 and a complementary sequence thereof, to determine the presence or amount of Haemophilus infl uenzae; and SEQ ID NO. 28, SEQ ID NO. 29 and a complementary sequence thereof, to determine the presence or quantity of Moraxella catarrhalis;
7. 7. The method of any of claims 1 to 6, which is performed directly on a sample obtained from human, animal, environmental or food patients.
8. 8. The method of any of the 15 claims 1 to 6, which is performed directly on a sample consisting of one or more bacterial colonies.
9. The method of any of claims 1 to 8, wherein the nucleic acids are amplified or detected by a method selected from the group 20 consisting of: a) polymerase chain reaction (PCR), b) ligase chain reaction, c) amplification based on the nucleic acid sequence, 25 d) self-sustained sequence replication, e) displacement amplification of chain, f) amplification of the branched DNA signal, and • g) Nested PCR.
10. The method of claim 9, wherein the nucleic acids are amplified by PCR.
11. 11. The method of claim 10, which achieves within an hour the determination of the presence of nucleic acids by performing, for each amplification cycle, an annealing step of only one second to • 10 55 ° C and a denaturing step of only one second at 95 ° C without any time specifically allowed for an elongation stage.
12. 12. A method as defined in any of claims 6 to 11, for detecting the presence or The amount of the bacterial species in a test sample comprising the following steps: a) treating the sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least twelve nucleotides of 20 length, being a preparer of the pair of preparators capable of selectively hybridizing with one of the two complementary strands of the bacterial species DNA and of any of one of the sequences to determine the presence or amount of the bacterial species that 25 contain an objective sequence, and the other preparer of the pair of preparators is able to hybridize with the other of the chains so as to form an extension product containing the target sequence as a standard; b) synthesizing an extension product from each of the preparers whose extension product contains the target sequence, and amplifying the target sequence, if any, to a perceptible level; and c) detecting the presence or amount of target sequence amplified as an indication of the presence or amount of the bacterial species in the test sample.
13. 13. A method as defined in any of claims 1 to 12, wherein the step of detecting the presence or amount of amplified target sequence is performed by hybridizing it to the probe.
14. The method of any of claims 6 to 13, wherein the amplification primers comprise at least one pair of primers selected from: SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 55 and SEQ ID NO: 56, and SEQ ID NO: 131 and SEQ ID NO: 132, for the detection of Esterichia coli; SEQ ID IsTO: 112 and SEQ ID NO: 113, SEQ ID NO: 118 and SEQ ID NO: 119, and SEQ ID NO: 160 and SEQ ID NO: 119 for the detection of Moraxella catarrhalis; SEQ ID NO: 83 and SEQ ID No. 84, and SEQ ID NO: 85 and SEQ ID NO: 86; for the detection of Pseudomonas aeruginosa; SEQ ID NO: 145 and SEQ ID NO: 146, and SEQ ID NO: 147 and SEQ ID NO: 148, for the detection of Staphylococcus epidermidis; SEQ ID NO: 149 and SEQ ID NO: 150, SEQ ID NO: 149 and SEQ ID NO: 151, and, SEQ ID NO: 152 and SEQ ID NO: 153, for the detection of Straphylococcus aureus; SEQ ID NO: 78 and SEQ ID NO: 79, SEQ ID NO: 156 and SEQ ID NO: 157, and SEQ ID NO: 158 and SEQ ID NO: 159; for the detection of Streptococcus pneumoniae; SEQ ID NO: 143 and SEQ NO: 144, for the detection of a bacterial species that carries the SpeA exotoxin A gene; SEQ ID NO: 141 and 142, for the detection of Streptococcus pyogenes; SEQ ID NO: 38 and SEQ ID NO: 39, and SEQ ID NO: 40 and SEQ ID NO: 41, for the detection of Enterococcus faecalis; SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 135 and SEQ ID NO: 136, and SEQ ID NO: 137 and SEQ ID NO: 138, for the detection of Klebsiella pneumoniae; SEQ ID NO: 74 and SEQ ID NO: 75, and SEQ ID NO: 133 and SEQ ID NO: 134, for the detection of Proteus mirabilis; SEQ NO: 98 and SEQ ID NO: 99, and SEQ NO: 139 and SEQ ID NO: 140, for the detection of Staphylococcus saprophyticus; or SEQ NO: 154 and SEQ ID NO: 155, for the detection of Haemophilus influenzae.
15. 15. The method of claim 13, wherein the probe is selected from: SEQ ID NO: 44, SEQ ID NO .: 45, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and a complementary sequence thereof for the detection of Escherichia coli; SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO .: 80, SEQ ID NO: 81, SEQ ID NO: 82, and a complementary sequence thereof for the detection of Proteus mirabilis; SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, and a complementary sequence thereof for the detection of Klebsiella pneumoniae; SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, and a complementary sequence thereof. the detection of Staphylococcus saprophyticus; SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, and one complementary sequence thereof for the detection of Moraxella catarrhalis; SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and a complementary sequence thereof for the detection of Pseudomonas aeruginosa; SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, and a complementary sequence thereof for the detection of Haemophilus influenzae; or SEQ ID NO: 120, SEQ ID NO: 121, and a complementary sequence thereof for the detection of Streptococcus pneumoniae.
16. 16. A method as defined in any of claims 3 to 15, wherein the amplification primers further comprise a pair of primers having at least twelve nucleotides in length capable of hybridizing with any one of the defined nucleotide sequences. in: SEQ ID NO. 161 or a complementary sequence thereof, for the detection of a bacterial resistance to β-lactam antibiotics mediated by the bacterial gene blatem of antibiotic resistance; SEQ ID No. 162 or a complementary sequence thereof, for the detection of a bacterial resistance to β-lactam antibiotics mediated by the bacterial gene blaCOb of antibiotic resistance; SEQ ID NO. 163 or a complementary sequence thereof, for the detection of a bacterial resistance to β-lactam antibiotics mediated by the bacterial gene blaShv of antibiotic resistance; SEQ 10 NO. 164 or a complementary sequence thereof, for the detection of a bacterial resistance to inoglycosides mediated by the bacterial gene aadB of antibiotic resistance; SEQ ID NO. 165 or a complementary sequence thereof, for the detection of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial gene aacCl of antibiotic resistance; SEQ ID NO. 166 or a complementary sequence thereof, for the detection of a bacterial resistance to aminoglycoside antibiotics. mediated by the aacC2 bacterial gene of antibiotic resistance; SEQ NO. 167 or a complementary sequence thereof, for the detection of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial gene aacC2 of antibiotic resistance; SEQ ID NO. 168 or a complementary sequence thereof, for the detection of a bacterial resistance to aminoglycoside antibiotics mediated by the aacCA bacterial gene of antibiotic resistance; SEQ ID NO. 169 or a complementary sequence thereof, for the detection of a bacterial resistance to ß-lactam antibiotics mediated by the gene. SEQ ID NO. 170 or a complementary sequence thereof, for the detection of a bacterial resistance to vancomycin mediated by the vanH, vanA and vanx bacterial genes of antibiotic resistance / SEQ ID NO. 173 or a complementary sequence thereof, for the detection of bacterial resistance to streptogramin A mediated by the bacterial gene satA of antibiotic resistance; SEQ ID NO. 174 or a complementary sequence thereof, for the detection of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial gene aacA-aphD of antibiotic resistance; SEQ ID NO. 175 or a complementary sequence thereof, for the detection of a bacterial resistance to virginiamycin mediated by the bacterial vat gene of antibiotic resistance; SEQ ID NO. 176 or a complementary sequence thereof, for the detection of a bacterial resistance to virginiamycin mediated by the bacterial gene vga of antibiotic resistance; SEQ ID NO. 177 or a complementary sequence thereof, for the detection of a bacterial resistance to erythromycin mediated by the bacterial gene msrA of antibiotic resistance; SEQ ID NO. 171 or a complementary sequence thereof, for the detection of a bacterial resistance to β-lactam, aminoglycosides, chloramphenicol or trimethoprim mediated by the bacterial gene int of antibiotic resistance; and SEQ ID NO. 172 or a complementary sequence thereof, for the detection of a bacterial resistance to ß-lactam antibiotics, of aminoglycosides, of chloramphenicol or trimethoprim mediated by the bacterial gene sul of antibiotic resistance.
17. 17. A method as defined in any of claims 13 to 16, further comprising a probe for detecting any bacterial species, the probe having at least one single-stranded nucleic acid having at least twelve nucleotides in length capable of hybridizing with any of the bacterial species of and with at least one of SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 and a complementary sequence thereof.
18. 18. A method as defined in claim 17, wherein the universal probe is selected from the group consisting of SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 130 and a complementary sequence thereof.
19. 19. A method as defined in any of claims 2 to 16, wherein the primers for detecting any bacterial species comprise at least one single-stranded nucleic acid whose nucleotide sequence is at least twelve nucleotides in length capable of hybridizing with any bacterial species and with at least one of SEQ ID NO: 128 and SEQ ID NO: 127 and a complementary sequence thereof.
20. 20. A method as defined in claim 19, wherein the preparers comprise SEQ ID NO: 126 and

    127.
21. 21. A nucleic acid that is at least twelve nucleotides in length and that is capable of hybridizing to the nucleotide sequence of any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9. SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO.13, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, and a complementary sequence thereof, when in the simple chain form, and which hybridizes ubiquitously and specifically, with its target bacterial DNA defined in claim 6 as a probe or as a preparer.
22. 22. An oligonucleotide having the nucleotide sequence of any of SEQ ID NOS: 38 to 125, and 127 to 160.
23. 23. A recombinant plasmid comprising a nucleic acid as defined in claim 21.
24. 24. A host cell recombinant that has been transformed by a recombinant plasmid as defined in claim 23.
25. 25. A non-recombinant human host cell as defined in claim 24, wherein the host cell is Esterichia coli.
26. 26. A diagnostic kit for the detection or quantification of nucleic acids of any combination of the bacterial species defined in any of claims 6 to 20 comprising any combination of preparer pairs, with or without probes, as defined in I presented.
27. 27. A diagnostic equipment as defined in claim 26, which further comprises any combination of trainers, with or without probes, as defined in any of claims 3 to 11 and 16 for the detection or quantification of the nucleic acids of any combination of the bacterial or toxin resistance genes, as defined herein.
28. 28. A diagnostic equipment for the detection or quantification of nucleic acids of any bacterial species comprising the preparations defined in SEQ NOS. 126 and 127.
29. 29. A diagnostic kit for the simultaneous detection or quantification of any bacterial species, of any antibiotic-resistant gene, and of any combination of bacterial species defined in any of claims 6 to 20 comprising any combination of preparer pairs defined in any of claims 13 to 20, a pair of universal preparers, and any combination of preparer pairs that specifically anneal to the antibiotic resistance genes defined in any of SEQ ID NOS: 161 to 177, Preparators are used with or without probes. SUMMARY OF THE INVENTION The present invention relates to methods, based on DNA, for universal bacterial detection, for the specific detection of common bacterial pathogens, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus. , Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and Moraxella ca tarrhalis, as well as for the specific detection of bacterial genes resistant to antibiotics, commonly found and clinically relevant, directly from clinical specimens or, alternatively, from a bacterial colony. The above bacterial species can account for as much as 80% of the bacterial pathogens isolated in the usual microbiology laboratories. The core of this invention consists primarily of the DNA sequences from all the genomic DNA fragments of specific species, selected by the hybridization of the genomic collections or, alternatively, selected from data banks, as well as any sequence of derived oligonucleotides. of these sequences, which can be used as probes or amplification primers for the polymerase chain reaction (PCR) or any other nucleic acid amplification method. This invention also includes DNA sequences of selected antibiotic resistance genes clinically relevant. With these methods, bacteria can be detected (universal preparers and / or probes) and identify (preparers and / or probes of specific species) directly from clinical specimens or from an isolated bacterial colony. Bacteria are also evaluated for their suspected susceptibility to antibiotics by the detection of resistance genes (specific preparations and / or probes resistant to antibiotics). Diagnostic equipment for the presence detection for the bacterial identification of bacterial species, mentioned above, and for the detection of antibiotic resistance genes, are also claimed. These devices for rapid (one hour or less) and accurate diagnosis of bacterial infections and resistance to antibiotics, will gradually replace the conventional methods currently used in clinical microbiology laboratories for routine diagnostics. They will provide tools to clinics to help prescribe timely optimal treatments when necessary. Consequently, these tests will contribute to saving human lives, rationalize treatments, reduce the development of antibiotic resistance and avoid unnecessary hospitalizations.