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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
  • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

• the present invention relates to the field of diagnostics. More specifically, the invention relates to a method for the molecular identification of bacteria of the genus Staphylococcus by detection and / or amplification and sequencing techniques using probes or oligonucleotide primers applied to strains of this genus bacterial.
• the bacteria of the genus Staphylococcus are cocciform, Gram-positive and catalase-positive bacteria of which 36 species are currently recognized, 9 of which include subspecies [Euzéby JP. (1997) Int J Syst Bacteriol 47: 590-2]. These species are coagulase-negative, with the exception of Staphylococcus aureus, Staphylococcus intermedius, Staphylococcus delphinii, Staphylococcus schleiferi subsp. coagulans, and some strains of Staphylococcus hyicus [Kloos WE (1995) In Manual of Clinical Microbiology, pp 282-298, ASM Press].
• Staphylococcus aureus is a coagulase-positive species responsible for food poisoning linked to the production of an enterotoxin, toxic staphylococcal shock, and purulent infections characterized by septic metastases distant from the initial infectious focus.
• the molecular targets currently proposed for the molecular identification of bacteria of the genus Staphylococcus include the 16S rDNA gene encoding the 16S subunit of ribosomal RNA [Bialkowska-Hobrzanska H et al. (1990) Eur. J. Microbiol. Infect. Dis. 9: 588-594], the spacer of the genes encoding the transfer RNAs [Maes N. et al. (1997) J. Clin. Microbiol. 35: 2477-2481], the hsp60 gene coding for the stress protein 60 [Goh SH et al. (1996) J. Clin. Microbiol. 34: 818-823; Goh SH (1997) J. Clin. Microbiol.
• Hybridization of oligonucleotides is the technique generally proposed for targeting these identification regions. Detection of the naked gene is limited to bacteria of the species Staphylococcus aureus [Brakstad OG (1992) J. Clin. Microbiol. 30: 1654-1660] and a chromosomal fragment has been reported for the identification of bacteria of the species Staphylococcus epidermidis [Martineau F (1996) J. Clin. Microbiol. 34: 2888-2893].
• the present invention relates to nucleic acid sequences specific for the genus or for each species of the genus Staphylococcus, the nucleotide sequence of which is taken from the rpoB gene of said bacteria.
• RNA polymerases are divided into two groups according to their origin, one consisting of the RNA- or DNA-dependent RNA polymerases, and the other consisting of the DNA-dependent RNA polymerases of eukaryotic or prokaryotic origin (archaebacteria and eubacteria).
• the eubacterial DNA-dependent RNA polymerases are characterized by a simple and conserved multimeric constitution noted "core enzyme", represented by ⁇ ', or "holoenzyme” represented by cc ⁇ ' ⁇ [Yura and Ishihama, Ann. Rev. Broom. (1979) 13: 59-97].
• the genes which code for the different ⁇ ' ⁇ subunits of DNA-dependent RNA polymerase in eubacteria are classified into different groups comprising the genes coding for proteins constituting the sub - ribosomal units or for enzymes involved in the replication and repair of the genome [Yura and Yshihma, Ann. Rev. Broom. (1979) 13: 59-97].
• Some authors have shown that the sequences of the rpoB and rpoC genes can be used to construct phylogenetic trees [Rowland et al. Biochem. Soc. Trans. (1992) 21: 40S] making it possible to separate the different branches and sub-branches among the kingdoms of the living.
• nucleic acid extracted from bacteria means either the total nucleic acid, or genomic DNA, or the messenger RNAs, or even the DNA obtained from the reverse transcription of the messenger RNAs;
• nucleotide fragment or an “oligonucleotide” are two synonymous terms designating a sequence of nucleotide motifs characterized by an informational sequence of natural (or possibly modified) nucleic acids and capable of hybridizing, like natural nucleic acids, with a complementary or substantially complementary nucleotide fragment, under predetermined conditions of strict stringency.
• the chain may contain nucleotide motifs with a structure different from that of natural nucleic acids.
• a nucleotide fragment (or oligonucleotide) can contain, for example, up to 100 nucleotide motifs.
• a nucleotide motif is derived from a monomer which can be a natural nucleotide of nucleic acid, the constituent elements of which are a sugar, a phosphate group and a nitrogenous base chosen from adenine (A), guanine (G), Puracil (U), cytosine ( C), thymine (T); or the monomer is a nucleotide modified in at least one of the three preceding constituent elements; by way of example, the modification can take place either at the level of the bases, with modified bases such as inosine, which can be hydrated with any base A, T, U, C or G, methyl-5-deoxycytidine , deoxyuridine, dimethylamino-5-deoxyuridine or any other modified base capable of hybridization, either at
• hybridization means the process during which, under appropriate conditions, two nucleotide fragments having sufficiently complementary sequences are capable of being combined by stable and specific hydrogen bonds, to form a double strand.
• the hybridization conditions are determined by "stringency", that is to say the rigor of the operating conditions. Hybridization is all the more specific as it is performed at higher stringency.
• the stringency is a function in particular of the base composition of a probe / target duplex, as well as by the degree of mismatch between two nucleic acids.
• the stringency can also be a function of the parameters of the hybridization reaction, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and / or the temperature of hybridization.
• the stringency of the conditions under which a hybridization reaction must be carried out depends in particular on the probes used. All these data are well known and the appropriate conditions can possibly be determined in each case by routine experiments. In general, depending on the length of the probes used, the temperature for the hybridization reaction is between about 20 and 65 ° C, in particular between 35 and 65 ° C in saline at a concentration of about 0.8 to 1 M.
• a “probe” is a nucleotide fragment having a specificity of hybridization under determined conditions to form a hybridization complex with a nucleic acid having, in the present case, a nucleotide sequence included either in a messenger RNA, or in a DNA obtained by reverse transcription of said messenger RNA, transcription product; a probe can be used for diagnostic purposes (in particular capture or detection probes) or for therapy purposes, - a “capture probe” is a probe immobilized or immobilizable on a solid support by any suitable means, for example by covalence, by adsorption, or by direct synthesis on a solid. Examples of carriers include microtiter plates and DNA chips,
• a “detection probe” is a probe marked by means of a marker agent chosen for example from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic, fluorigenic or luminescent substrate (in particular a peroxidase or alkaline phosphatase), chromophoric chemical compounds, chromogenic compounds, fluorigenic or luminescent, analogs of nucleotide bases and ligands such as biotin,
• a marker agent chosen for example from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic, fluorigenic or luminescent substrate (in particular a peroxidase or alkaline phosphatase), chromophoric chemical compounds, chromogenic compounds, fluorigenic or luminescent, analogs of nucleotide bases and ligands such as biotin,
• a "species probe” is a probe allowing the specific identification of the species of a bacterium
• a "genus probe” is a probe allowing the specific identification of the genus of a bacterium
• a “primer” is a probe comprising for example 10 to 100 nucleotide units and having a specificity of hybridization under conditions determined for the enzymatic amplification reactions
• amplification reaction means an enzymatic polymerization reaction , for example in an amplification technique such as PCR, initiated by priming oligonucleotides and using DNA polymerase.
• “sequencing reaction” means obtaining the sequence of a nucleic acid fragment or a complete gene by an abortive polymerization process from oligonucleotide primers and using said dideoxynucleotides (Sanger F, Coulson AR (1975), J. Mol. Biol. 94: 441) or by multiple hybridizations with multiple probes fixed on solid support as used in DNA chips for example.
• the inventors have determined the complete sequences of the rpoB genes of four species of bacteria of the genus Staphylococcus. These four species have been chosen by the inventors as representing the four main genetic groups determined on the basis of the study of the 16S gene in bacteria of the genus Staphylococcus, namely the phylogenetically most divergent species among all the species currently described. in this genus, so that the alignment of the rpoB sequences obtained in these four species can probably phylogenetically frame all of the rpoB sequences of all the species of this bacterial genus.
• the inventors have highlighted the consensus and specific sequences SEQ.ID. n ° 7 to 10 described in the sequence listing at the end of the description. The inventors determined said sequences SEQ.ID. n ° 7 to 10 as being not only consensual between all bacteria of the genus
• Staphylococcus but also specific to the family of bacteria of the genus Staphylococcus, except Staphylococcus schleiferi with regard to the sequence SEQ.ID. # 8.
• sequences are present in the rpoB genes of any bacteria of the genus Staphylococcus and specific to bacteria of the genus Staphylococcus which can be used as a genes probe to detect any bacterium of the genus Staphylococcus except Staphylococcus schleiferi as regards the sequence SEQ.ID. # 8.
• nucleotide N mentioned in the sequence listing at the end of the description can represent inosine or an equimolar mixture of 4 different nucleotides chosen from A, T, C and Gt, or A, U, C respectively and G, insofar as, as mentioned in the definitions, an oligonucleotide or a nucleic acid fragment according to the invention may be in the form of an oxyribonucleic acid (DNA) or a ribonucleic acid (RNA) for which, in this case, T is replaced by U.
• DNA oxyribonucleic acid
• RNA ribonucleic acid
• N represents a said equimolar mixture of nucleotides at a given position
• the nucleotide at said given position indifferently represents A, T, C or G (or respectively the where appropriate A, U, C or G) and that the oligonucleotide according to the invention consists more precisely of an equimolar mixture of 4 groups of oligonucleotides in each of which groups N has a different meaning at said position given and represented each of the 4 bases A, T, C or G (or A, U, C or G respectively).
• variable nucleotides are found in the complementary target sequences according to the species of the bacteria considered, but all the other nucleotides are conserved in all the species of bacteria of the genus Staphylococcus. Because "N” represents inosine which can hybridize with any base or an equimolar mixture of the 4 bases A, T, C, G, the sequences SEQ.ID. No. 7 and 10 can hybridize with the complementary sequence included in the rpoB gene of all bacteria of the genus Staphylococcus.
• consensus sequences SEQ.ID. n ° 9 and SEQ ID n ° 10 surround hypervariable sequences whose sequence is specific for each species of bacteria of the genus Staphylococcus. Sequences framed by SEQ.ID. No. 9 and 10 can therefore be used as a species probe for bacteria of the genus Staphylococcus.
• sequences SEQ.ID. Nos. 9 and 10 were determined to frame a fragment of the rpoB gene comprising an area whose variable length is approximately 500 bp and constitutes the shortest specific sequence for each species of the bacterium of the genus Staphylococcus.
• the inventors were thus able to highlight species probes for each of the 29 species of bacteria of the genus Staphylococcus studied corresponding to the sequences SEQ.ID. n ° 11 to 39 framed by the SEQ.ID consensus sequences. n ° 9 and 10.
• the consensus sequences SEQ.ID. No. 7 to 10 identified according to the present invention can be used as an amplification primer or a sequencing reaction in methods for detecting bacteria of the genus Staphylococcus by molecular identification.
• the SEQ.ID. n ° 7 to 10 therefore make it possible not only to prepare genus probes for bacteria of the genus Staphylococcus but also to detect and identify the species of said bacterium by amplification and sequencing using said sequences as primers. More specifically, the present invention provides a method of detection by identification of a bacterium from one of the species of the genus Staphylococcus, characterized in that:
• the rpoB gene of said bacteria or a fragment of said rpoB gene of said bacteria comprising a nucleotide sequence chosen from one of the sequences SEQ.ID Nos. 11 to 29 and 31 to 39, the reverse sequences and the complementary sequences, or
• a fragment of said rpoB gene of said bacterium consisting of the nucleotide sequence SEQ.ID No. 30, the reverse sequence and the complementary sequence, or - an oligonucleotide comprising a sequence of at least 12 consecutive nucleotide motifs, included in the one of the sequences SEQ.ID. n ° 7 to 10, in which N represents a nucleotide chosen from inosine or a equimolar mixture of 4 different nucleotides chosen from A, T, C or G, the reverse sequences and the complementary sequences.
• Said oligonucleotides preferably comprise from 12 to 35 nucleotide units, and more preferably, said oligonucleotides consist of the sequences SEQ.ID. n ° 7 to 10, the reverse sequences and the complementary sequences.
• the amplification primers comprising said oligonucleotides comprising a sequence of at least 12 nucleotide motifs included in at least two sequences drawn from the sequences SEQ.ID are brought into contact.
• n ° 7 to 10 reverse sequences and complementary sequences, with a sample containing or likely to contain nucleic acids of at least one such bacterium of the genus Staphylococcus, with:
• an oligonucleotide chosen from oligonucleotides comprising a sequence included in one of the sequences SEQ.ID. No. 7 or 9 or the complementary sequences, preferably an oligonucleotide consisting of the said complete sequences, and
• an oligonucleotide comprising a sequence included in one of the sequences SEQ.ID. n ° 10 or 8 or respectively a sequence complementary, preferably an oligonucleotide consisting of said complete sequences.
• an amplification of nucleic acids is carried out by enzymatic polymerization reaction and the appearance or absence of an amplification product is determined, and the presence of said bacteria in the sample is thus determined if a product d amplification appeared.
• an oligonucleotide of sequence SEQ.ID # 7 or 9 or a complementary sequence is used as primer 5 "and an oligonucleotide of sequence SEQ.ID.n ° 10 or respectively a complementary sequence.
• a bacterium of the genus Staphylococcus chosen from the species Staphylococcus xylosus, Staphylococcus warneri, Staphylococcus simulans, Staphylococcus sciuri, Staphylococcus schleiferi, Staphylococcus saphrophyticus, Staphylococcus saccharolyticus, Staphylococcus pulveris, Staphylococcus muscae, Staphylococcus lugdunensis, Staphylococcus lentis, Staphylococcus kloosii, Staphylococcus intermedius, Staphylococcus hyicus, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus gallinarum, Staphylococcus felis Staphylococcus
• a sample containing or likely to contain nucleic acids from at least one such bacterium is brought into contact with at least one species probe consisting of a said gene fragment comprising a sequence included in one of the sequences SEQ .ID. Nos. 11 to 39, the reverse sequences and complementary sequences, preferably an oligonucleotide consisting of one of said sequences SEQ.ID. n ° 11 to 39, or an oligonucleotide of reverse or complementary sequence, and
• the method comprises the steps in which, in a sample containing or likely to contain nucleic acids from at least one said bacterium: a) a sequencing reaction of a fragment of the amplified rpoB gene of a said given bacterium is carried out using the nucleotide primers consisting of oligonucleotides comprising a sequence included in the sequences SEQ.ID.
• step a) the steps are carried out comprising:
• 2- a sequencing reaction of the amplifiers determined in step 1 with the primers 5 'and 3' consisting of oligonucleotides comprising the sequences SEQ.ID. n ° 9 and respectively SEQ.ID. # 10, preferably consisting of said sequences SEQ.ID. Nos. 7 and 10 or their complementary sequences, preferably consisting of the said sequences SEQ.ID. n ° 9 and 10 or their complementary sequences, and
• step b) the sequences obtained are compared with one of the sequences SEQ.ID respectively. n ° 1 1 to 39 or their complementary sequences.
• Another object of the present invention is a rpoB gene or gene fragment of a bacterium of the genus Staphylococcus, characterized in that it comprises a sequence as described in the sequences SEQ.ID. n ° 11 to 29 and 30 to
• Another subject of the present invention is the complete sequence of the rpoB gene of the bacteria Staphylococcus saccharolyticus, Staphylococcus lugdunensis, Staphylococcus caprae, and Staphylococcus intermedius as described in the sequences SEQ.ID. n ° 3 to 6, as mentioned previously, these fragments of rpoB genes and complete genes are useful in particular for a method according to the invention.
• the complete sequence of the rpoB gene can be used to identify the bacterium not only by studying its primary sequence, but also, by studying the secondary and tertiary structures of messenger RNA originating from the transcription of the complete sequence d DNA.
• Another object of the present invention is a so-called rpoB gene fragment or oligonucleotide chosen from oligonucleotides having a sequence consisting of sequences SEQ ID No. 1 1 to 39 and from oligonucleotides of reverse sequences and complementary sequences as defined above above.
• Another object of the present invention is an oligonucleotide comprising a sequence of at least 12, preferably 12 to 35, consecutive nucleotide motifs included in one of the sequences SEQ.ID.
• N represents a nucleotide chosen from inosine and an equimolar mixture of 4 different nucleotides chosen from A, T, C or G, and the oligonucleotides of reverse sequences and complementary sequences, preferably consisting of SEQ.ID sequences n ° 7 and 10 and the reverse sequences and complementary sequences in which N represents inosine.
• the sequences SEQ ID No. 7 to 39 can be prepared by chemical synthesis using the techniques well known to those skilled in the art and described for example in the article by Itakura K. et al. [(1984) Annu. Rev. Biochem. 53: 323].
• a first application of an oligonucleotide according to the invention is its use as a probe for the detection, in a biological sample, of bacteria of one of the species of the genus Staphylococcus which comprises a nucleotide sequence of at least 12 consecutive nucleotide motifs included in one of the sequences SEQ ID No. 7 to 39, and their reverse or complementary sequences.
• a probe comprising the sequences SEQ.ID. No. 7 to 10 will be used as a gender probe and a probe comprising one of the sequences SEQ.ID. n ° 1 1 to 39 will be used as a species probe.
• the probes according to the invention can be used, for diagnostic purposes, as mentioned above, by determining the formation or absence of formation of a hybridization complex between the probe and a target nucleic acid in a sample, according to all the known hybridization techniques and in particular the point deposition techniques on a filter, called "DOT-BLOT” [Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor], so-called “SOUTHERN BLOT” DNA transfer techniques [Southern EM, J. Mol. Biol.
• the nucleic acid to be detected can be DNA or RNA (the first obtained after amplification by PCR).
• the target nucleic acid can be obtained by extraction, according to known methods, from the nucleic acids of a sample to be examined.
• the denaturation of a double-stranded nucleic acid can be carried out by known methods of chemical, physical or enzymatic denaturation, and in particular by heating to an appropriate temperature, above 80 ° C.
• a probe of the invention called the capture probe is immobilized on a solid support, and another probe of the invention, called the detection, is labeled with a labeling agent.
• a labeling agent is labeled with support and of labeling agent.
• a species probe is immobilized on a solid support, and another species probe is labeled with a labeling agent.
• Another application of an oligonucleotide of the invention is its use as a nucleotide primer comprising a single-stranded oligonucleotide chosen from oligonucleotides having a sequence of at least 12 nucleotide motifs included in one of the sequences SEQ ID Nos. 7 to 39, which can be used in the synthesis of a nucleic acid in the presence of a polymerase by a process known per se, in particular in amplification methods using such a synthesis in the presence of a polymerase (PCR, RT-PCR, etc. ).
• a primer of the invention can be used for the specific reverse transcription of a messenger RNA sequence of bacteria of a species of the genus Staphylococcus to obtain a corresponding complementary DNA sequence.
• a reverse transcription can constitute the first stage of the RT-PCR technique, the next stage being the amplification by PCR of the complementary DNA obtained.
• the primers of the invention can also be used for the specific amplification by polymerase chain reaction of the total DNA sequence of the rpoB gene of a species of the genus Staphylococcus.
• said primer comprising an oligonucleotide of the invention further comprises the sense or antisense sequence of a promoter recognized by an RNA polymerase (promoters T7, T3, SP6 for example [Studier FW, BA Moffatt (1986) J. Mol. Biol. 189: 1 13]: such primers are usable in nucleic acid amplification methods involving a transcription step, such as, for example, the NASBA or 3SR techniques [Van Gemen B. et al. Abstract MA 1091, 7 th International Conference on AIDS (1991) Florence, Italy].
• nucleotide primer comprising a single-stranded oligonucleotide chosen from oligonucleotides having a sequence comprising one of the sequences SEQ ID No. 11 to 29 and 31 to 39, or preferably consisting of one of SEQ.ID sequences n ° 11 to 39 which can be used for the total or partial sequencing of the rpoB gene of any strain of a species of the genus Staphylococcus.
• the sequencing of the partial or complete rpoB gene in any bacteria of the genus Staphylococcus allows the identification of any Staphylococcus bacteria by bioinformatic analysis of this sequence and the recognition of new species of unknown Staphylococcus bacteria.
• the sequences SEQ ID No. 7 to SEQ ID No. 10 are used, in which N is inosine preferably, the sequences SEQ ID No. 7 and SEQ .ID. # 10.
• the present invention also relates to a diagnostic kit useful in a method according to the invention comprising at least one said gene fragment of said oligonucleotide consisting of the sequences SEQ.ID. No. 7 to 39 and the reverse sequences and complementary sequences or a said oligonucleotide comprising a sequence included in one of the sequences SEQ.ID. n ° 7 to 10, and / or at least one said rpoB gene fragment of a said bacterium comprising the sequences SEQ.ID. nos. 11 to 39, and the oligonucleotides and gene fragments of reverse sequences and complementary sequences, as defined above.
• reverse sequences and complementary sequences means the following sequences:
• a final object of the invention is a gene therapy probe for treating infections caused by a strain belonging to a species. of the genus Staphylococcus, said probe comprising an oligonucleotide as defined above.
• This gene therapy probe capable of hybridizing to messenger RNA and / or to the genomic DNA of said bacteria, can block the phenomena of translation and / or transcription and / or replication.
• the principle of gene therapy methods is known and is based in particular on the use of a probe corresponding to an antisense strand: the formation of a hybrid between the probe and the sense strand is capable of disturbing at least one stages of deciphering genetic information.
• Gene therapy probes can therefore be used as antibacterial drugs, making it possible to fight against infections caused by bacteria of species of the genus Staphylococcus.
• FIG. 1 represents the visualization of the amplification products by staining with ethidium bromide after electrophoresis on an agarose gel obtained in Example 3.
• Example 1 Sequence of the rpoB gene of four species of the genus Staphylococcus: Staphylococcus saccharolyticus, Staphylococcus lugdunensis, Staphylococcus caprae and Staphylococcus intermedius.
• the complete sequence of the rpoB gene of bacteria of the species Staphylococcus saccharolyticus, Staphylococcus lugdunensis, Staphylococcus caprae and Staphylococcus intermedius was determined by enzymatic amplification and direct automatic sequencing using primers consensus between the sequences of the rpoB gene in Staphylococcus aureus (GenBank n ° access X64172) Bacillus subtillis (GenBank access number L43593). The latter bacterial species was chosen as the Gram-positive species of low guanosine plus cytosine content closest to the species of the genus Staphylococcus (phylogenetic proximity based on the comparison of the 16S rDNA gene sequences).
• Several potential consensus primers have been investigated to obtain a fragment capable of leading to the complete sequence of rpoB genes by successive extensions from a series of specific primers. These consensus primers have the following sequences:
• the amplifications were carried out under a final volume of 50 ⁇ l comprising 2.5 ⁇ 10 ⁇ 2 2 U of Taq polymerase, 1 X of Taq buffer and 1.8 mM of MgCl 2 , 200 ⁇ M of dATP, dTTP, dGTP, dCTP and 0.2 ⁇ M of each primer They were carried out according to the following program: 35 cycles comprising a denaturation step at 94 ° C for 30 seconds, hybridization of the primers at 52 ° C for 30 seconds and extension at 72 ° C for 60 seconds
• the amplification products were purified on a column and then sequenced using the oligonucleotide sequencing primers presented in the following table: 0
• the sequencing reactions were carried out using the reagents of the ABI Prism dRhodamine Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems) kit according to the supplier's recommendations according to the following program: 30 cycles including a denaturation step at 94 ° C for 10 sec, a step of hybridization of the primer at 50 ° C for 10 sec. and an extension step at 60 ° C for 2 minutes.
• the sequencing products were separated by electrophoresis on a polyacrylamide gel on a 377 DNA Sequencer (Perkin) and analyzed to form 0 consensus sequences by the Sequence Assembler software (Applied Biosystems). This approach allowed us to determine the complete sequence of the rpoB gene in four species of the genus Staphylococcus:
• SEQ ID N ° 3 Sequence of the rpoB gene of Staphylococcus 5 saccharolyticus. This sequence measures 3,791 base pairs and has a cytosine plus guanosine content of 36.8% and is deposited in GenBank under the number GenBank accession AF325871.
• SEQ ID N ° 4 Sequence of the rapB gene of Staphylococcus lugdunensis This sequence measures 3,855 base pairs and has a cytosine plus guanosine content of 36.4% is deposited in GenBank under the number GenBank accession AF325870.
• SEQ ID N ° 5 Sequence of the rpoB gene of Staphylococcus caprae This sequence measures 3,698 base pairs and has a cytosine plus guanosine content of 37.4% is deposited in GenBank under the number GenBank accession AF325868.
• SEQ ID N ° 6 Sequence of the rpoB gene of Staphylococcus intermedius This sequence measures 3.851 base pairs and has a cytosine plus guanosine content of 39.2%, it is deposited in GenBank under the number GenBank accession AF325869).
• This sequence measures 3.852 base pairs and has a cytosine plus guanosine content of 39.2%, it is deposited in GenBank under the number GenBank accession AF325869.
• Example 2 Partial sequencing of the rpoB gene of 26 species of the genus Staphylococcus.
• the alignment of the rpoB sequence determined in the bacteria of the species Staphylococcus aureus, Staphylococcus lugdunensis (GenBank accession AF325870), Staphylococcus intermedius (GenBank accession AF325869), Staphylococcus saccharolyticus (GenBank accession AF325871) and Staphyl3bocankus accession) determine the consensus sequences of the following oligonucleotides positioned respectively in positions 2491-2511 and 3554-3573 of the rpoB gene in Staphylococcus aureus:
• SEQ ID No. 8 5 '- CCGTCCCAAGTCATGAAAC - 3' theoretically determining the amplification of a fragment of 1,063 base pairs in any species of the genus Staphylococcus.
• SEQ.ID. No. 8 is used as a 3 ′ primer and therefore represents the reverse sequence complementary to the direct strand represented in the sequences SEQ.ID. n ° 3 to 6 in position 3554-3573 in Staphylococcus aureus.
• the inventors determined the position of these two primers SEQ.ID. n ° 7 and SEQ.ID. n ° 8, in order to respect the following criteria:
• the inventors therefore by trial and error determined a new oligonucleotide of sequence SEQ ID No. 10 positioned at position 3241-3261 in Staphylococcus aureus, which combined with the oligonucleotide SEQ ID No. 7 in a PCR amplification reaction, has effectively made it possible to '' obtain an amplicon of the rpoB gene with a size of 771 base pairs (size for the species Staphylococcus aureus) in the 29 species of the genus Staphylococcus tested by the inventors.
• SEQ ID n ° 10 5'- GCIACITG ITCCATACCTGT - 3 'SEQ.ID. # 10 is used as a 3 'primer. This is why it corresponds to the reverse sequence complementary to the sequences of the direct strand represented on the sequences SEQ.ID. n ° 3 to 6.
• This last primer was determined to respect the constraints of a sequencing primer, that is to say of a size greater than 15 mothers, not hybridizing with the second primer used for sequencing, and framing a zone d generally about 500 base pairs whose sequence is specific for each species in the genus Staphylococcus.
• the inventors were therefore finally able to determine the partial sequence of the rpoB gene in 29 species of the genus Staphylococcus presented below (SEQ ID No. 11 to SEQ ID No. 39).
• the fragment of the rpoB gene was amplified by the PCR technique using 35 amplification cycles each comprising a denaturation phase of 94 ° C for 10 seconds, a phase of hybridization of the primers SEQ ID No. 7 and 8 or SEQ ID n ° 7 and 10 at 52 ° C for 20 seconds and an extension phase at 72 ° C for 60 seconds.
• the amplification product is visualized after coloring with ethidium bromide.
• the bacteria representing these 29 species of the genus Staphylococcus are as follows:
• the fragments in general about 500 base pairs of the rpoB gene of bacteria of species of the genus Staphylococcus, the sequence of which is specific for each species of this genus and therefore allowing the molecular identification of the bacteria of the 29 species tested are: SEQ ID N ° 11: Partial sequence of the rpoB gene in Staphylococcus xylosus, measuring 518 base pairs:
• SEQ ID N ° 12 Partial sequence of the rpoB Staphylococcus warneri gene measuring 507 base pairs:
• SEQ ID NO 13 Partial sequence of gene rpoB Staphylococcus simulans, measuring 518 base pairs: 5'TTCAGGGTTCATCAATGGTACTGCTTGACGTTGCATGTTCGCACCCATTAA CGCACGGTTAGAGTCATCGTTTTCTAAGAATGGGATACATGCTGTCGCTGC AGATACAACTTGTTTAGGAGAAACGTCCATATAGTCCATTTTCTCTCTATCCA TAGTTGTGTTGTTACCACGGAAACGACAAACGATTTCTTCGTCTAAGAAACG ACCTTCGTCATCTAAACGTGAGTTCGCTTGCGCACCGCAACAACATAGCTGTCTTCT TCGTCTGCAGTAAGGTAATCGATTTGATCTGTTACCGCATTTTTCTCATGGT CAACTTTACGATATGGTGTTTCAATGAAACCAAATTCATTAACACGCGCATAA CTTGATAATGAGTTGATTAAACCGATGTTCGGACCCTGGTGTCTCGATTG GACACATACGGCCATAGTGAGAGTAATGCACGTCACGT
• SEQ ID N ° 14 Partial sequence of the rpoB gene in Staphylococcus sciuri, measuring 507 base pairs:
• SEQ ID NO 15 Partial sequence of the rpoB gene in Staphylococcus schleiferi, measuring 518 base pairs: 5TTCTGGGTTTAACAATGGTACTGCTTGACGTTGCATGTTCGCACCCATCAA TGCACGGTTAGAGTCATCGTTTTCTAAAAACGGAATACATGCTGTCGCAGCT GAAACAACTTGTTTAGGCGATACGTCCATGTAGTCCATTTTTTCTTTAGCCAT AGTTGTGTTGTTACCACGGAAACGACAAACGATTTCGTCATCGATAAAACGT CCGTTTTCATCAAGTCTTGAGTTCGCTTGGGCAACAACATAACTGTCTTCTT CATCAGCAGTAAGGTAATCAATACGGTCTGTAATTGTGTTTGTTTCAAGGTC TACTTTTCTGTATGGAGTTTCAATGAAACCAAATTCATTCACACGTGCATAAC TTGAAAGTGAGTTGATCAAACCAATGTTTGGACCCTCTGGTGTCTCGATTGG ACACATACGGCCATAGTGAGAATAGTGTACG
• SEQ ID N ° 17 Partial sequence of the rpoB gene in Staphylococcus saccharolyticus, measuring 556 base pairs:
• SEQ ID NO 18 Partial sequence of the rpoB gene in Staphylococcus pulveris, measuring 508 base pairs: 5TTCAGGATTCATTAAAGGCACTGCTTGACGTTGCATGTTTGCACCCATAAG CGCACGGTTAGAGTCATCGTTTTCTAAGAAAGGAATACATGCTGTCGCAGC AGAAACAACCTGTTTAGGTGATACATCCATGTAATCCATACGTTCTTTAGGTT TCGTAGTATTATCCCCACGGAAACGACAAAGTACTTCATCATCATCAACGAATTT ACCTGTTTCATCAAGTACTGAGTTTGCTTGCGCTACAACATAGCTGTCTTCT TCGTCAGCTGTTAAATAGTCAATTCTGTCAGTAACTTGGTTTGTTTCGATATT AACCTTACGATAAGGCGTTTCAATAAAACCAAATTCATTAACTCTCGCATAAC TTGATAAAGAGTTAATTAAACCAAATTCATTAACTCTCGCATAAC TTGATAAAGAGTTAATTAAACCAAATGTTTGGTCCCTCAGGTTTGTTTCG
• SEQ ID NO 19 Partial sequence of the rpoB gene in Staphylococcus muscae, measuring 518 base pairs: 5TTCAGGATTCAACAATGGCACCGCTTGACGTTGCATGTTCGCACCCATTAA GGCACGGTTAGAGTCATCGTTTTCTAAGAATGGAATACATGCTGTCGCAGC AGAAACAACTTGCTTCGGCGATACGTCCATGTAGTCCATTTTCTCTTTTGCC ATTGTTGTGTTGTTACCACGGAAACGACATACAATCTCATCATCAATAAAGC GACCATTTTCATCTAAACGTGAGTTCGCTTGTGCAACCACATAACTATCTTCT TCATCAGCAGTTAAATAGTCGATTTGATCAGTGATTGTGTTCGTCTCGATAT CAACTTTACGATATGGTGTTTCAATGAAACCAAATTCATTAACACGTGCATAA CTAGATAGTGAGTTGATCAAACCAATGTTCAGTCCCTCTGGTGTCTCAATCG GACACATACGACCATAGTGAGAGTAGTGAAACCAATGTTCAGTC
• SEQ ID NO 20 Partial sequence of the rpoB gene in Staphylococcus lugdunensis, measuring 556 base pairs: 5TAACCCATTAGCAGAATTAACACACAAACGTCGTTTATCTGCGTTAGGACC TGGTGGTTTAACACGTGAACGTGCACAAATGGAAGTTCGTGACGTGCATTA TTCTCACTATGGCCGTATGTGTCCGATTGAAACACCAGAGGGTCCAAACATT GGTTTGATTAACTCATTATCTAGTTATGCGCGTGTCAACGAGTTTGGCTTTAT TGAAACGCCTTATCGTAAAGTAGATATTGATACAAATGCAATCACAGATCAA ATTGACTACTTAACTGCTGATGAAGAAGACAGTTATGTCGTTGCACAAGCGA ACTCTCGCCTTGATGAAAATGGTCGTTTCTTAGATGATGAAGTAGTATGCCG TTTCCGCGGTAATAATACTGTTATGGCTAAAGAAAATGGACTACATGGAT GTATCCTAAACAAGTTGG
• SEQ ID N ° 21 Partial sequence of the rpoB gene in Staphylococcus lentus, measuring 507 base pairs:
• SEQ ID NO 22 Partial sequence of the rpoB gene in Staphylococcus kloosii, measuring 505 base pairs: 5TTCACGGTTCATCAATGGTACCGCTTGACGTTGCATGTTCGCACCCATTAA GGCACGGTTAGAGTCATCGTTTTCTAAGAAAGGAATACATGCTGTCGCAGC CGAAACAACTTGTTTTGGTGATACGTCCATGTAGTCCATTTTTTCTTTCGCCA TAACTGTGTTGTTACCACGGAAACGACAAACTACTTCATCATCTAAGAAACG ACCATTTTCATCTAATTTAGAGTTAGCTTGCGCTACCACATAGCTATCTTCTT CATCAGCTGTTAAATAGTCAATTTGATCTGTGATTGAATTAGTTTCTAAATCA ACTTTACGGTATGGTGTTTCGATAAAGCCAAATTCATTAACACGTGCATAAC TTGATAATGAGTTGATAAGTCCAATGTTTGGACCCTCTGGCGTTTCGATTGG ACACATACGACCATAGTGAGAATAGTAACGTCAC
• SEQ ID N ° 23 Partial sequence of the rpoB gene in Staphylococcus intermedius, measuring 556 base pairs:
• SEQ ID N ° 24 Partial sequence of the rpoB gene in Staphylococcus hyicus, measuring 518 base pairs:
• SEQ ID N ° 25 Partial sequence of the rpoB gene in Staphylococcus hominis, measuring 518 base pairs:
• SEQ ID N ° 26 Partial sequence of the rpoB gene in Staphylococcus haemolyticus, measuring 507 base pairs:
• SEQ ID NO 27 Partial sequence of the rpoB gene in Staphylococcus gallinarum, measuring 507 base pairs: 5TTCAGGATTCATCAAAGGTACAGCTTGACGTTGCATGTTCGCACCCATCAA TGCACGGTTAGAGTCATCGTTTTCTAAGAAAGGAATACATGCTGTCGCAGCA GATACAACCTGTTTAGGTGATACATCCATGTAGTCCATTTTTTCTTTTGCCAT TACAGTGTTGTTACCACGGAAACGACAAACGACTTCATCTTCTACGAAACGA CCATTTTCATCTAATACAGAGTTTGCTTGTGCTACTACATAACTGTCTTCTTC ATCAGCTGTTAAGTAGTCAATTTGATCTGTAATAGATTGTGTTTCAATATCAA CTTTACGATATGGTGTTTCAATGAAACCAAATTCATTTACACGCGCATAACTT GATAATGAGTTGATAAGTCCGATGTTTGGACCCTCAGGTGTTTCGATTGGAC ACATACGGCCATAGTGAATAGTGAATAGTGAACGTCACGT
• SEQ ID NO 30 Partial sequence of the rpoB gene in Staphylococcus epidermidis, measuring 518 base pairs: 5TTCAGGATTCATTAAAGGCACCGCTTGACGTTGCATGTTTGCTCCCATTAA CGCACGGTTAGAGTCGTCATTTTCTAAGAATGGAATACATGCTGTTGCTGCT GAAACAACTTGTTTTGGTGATACGTCCATGTAATCCATTTTTTCTTTAGCCAT AACAGTGTTATTACCACGGAAACGACAAACAACTTCATCATCTAAGAAACGA CCATTCATCAAGTCTAGAATTAGCCTGTGCAACAACGTAGCTATCCTCTT CATCAGCTGTCAAATAATCTATTTGATCAGTGATTGAGTTTGTATCTAAATCC ACTTTACGATATGGCGTTTCAATAAAACCAAATTCATTCACTCTAGCATAACT TGACAATGAGTTTATTAAACCAATATTAGGACCCTCAGGTGTTTCAATTGGA CACATACGTGGCATTCCATTCACTCTAGCATAACT TGACAATGAGT
• SEQ ID NO 31 Partial sequence of the rpoB gene in Staphylococcus cohnii, measuring 507 base pairs: 5'TTCTGGATTCATCAATGGGACTGCTTGACGTTGCATGTTCGCACCCATTAA TGCACGGTTAGAGTCATCGTTTTCTAAGAATGGAATACATGCTGTTGCTGCA GAAACAACCTGTTTAGGAGATACATCCATGTAATCCATTTTTTCTTTTGCCAT AACTGTGTTATTACCACGGAAACGACAAACAACTTCATCATCTAAGAAGCGA CCATTTTCATCTAACTTAGAATTTGCTTGTGCTACTACATAGCTATCTTCTTC GTCAGCTGTTAAATAATCAATTTGATCTGTGATACTATTCGTTTCAAGATCTA CTTTACGATATGGCGTTTCAATGAAACCAAATTCATTTACACGTGCATAACTT GATAATGAGTTAATCAAACCAATGTTTGGTCCCTCTGGTGTTTCGATTGGACACACAC
• SEQ ID N ° 32 Partial sequence of the rpoB gene in Staphylococcus chromogenes, measuring 507 base pairs:
• SEQ ID N ° 34 Partial sequence of the rpoB gene in Staphylococcus capitis, measuring 518 base pairs:
• SEQ ID N ° 35 Partial sequence of the rpoB gene in Staphylococcus auricularis, measuring 507 base pairs:
• SEQ ID NO 36 Partial sequence of the rpoB gene in Staphylococcus aureus, measuring 518 base pairs: 5TTCTGGATTCATCAAAGGCACTGCTTGACGTTGCATGTTCGCACCCATCAA TGCACGGTTTGAGTCATCATTTTCTAAGAATGGAATACATGCTGTCGCTGCT GAAACAACTTGCTTCGGCGATACATCCATATAATCCATTTTTTCTTTAGCCAT AACTGTGTTGTTACCACGGAAACGACATACAACTTCATCATCATCCATGAAACGA CCATTTTCATCTAATTTAGAGTTTGCTTGTGCTACAACATAGCTATCTTCTTC GTCAGCTGTTAAATAGTCAATTTGATCAGTGATAGCATGTGTATCTAAATCAA CTTTACGATATGGTGTTTCAATAAAGCCGAATTCATTTACACGTGCATAACTT GATAATGAGTTAATCAATCCAATGTTTGGTCCCTCAGGTGTTTCAATTGGAC ACATACGGCCATAGTGAGTAGTGAACGTCACGTACTTCCAT
• SEQ ID N ° 38 Partial sequence of the rpoB gene in Staphylococcus arlettae, measuring 518 base pairs:
• SEQ ID N ° 39 Partial sequence of the rpoB gene in Staphylococcus caprae, measuring 556 base pairs:
• Example 3 Blind identification of a collection of 20 bacterial strains comprising 10 strains of bacteria belonging to the genus Staphylococcus.
• strains belonging to the following bacterial species Staphylococcus aureus (strain sensitive to rifampicin), Staphylococcus aureus (strain resistant to rifampicin), Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus Staphylococcus Staphylocus lugdunensis, Staphylococcus gallinarum, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Enterococcus faecalis, Steptococcus pyogenes, Corynebacterium amycolatum, Gemella morbilorum, Acinetobacter anitratus, aceticibular acupuncture and Micrococcus luteusea Acionibacter luteusea and Propionibea luteusea not knowing a priori the identity of the strains) of the strain
• the extraction of the nucleic acids as well as the amplification of the 751 base pair fragment of the rpoB gene were carried out as described in Example No. 2 by incorporating the primers SEQ ID No. 7 (as primer 5 ') and SEQ ID No. 10 (as 3 'primer) in PCR amplification (Fig. 1).
• the sequencing of these 10 amplifiers was carried out by incorporating into the sequencing reaction the primers SEQ ID No. 9 (primer 5 ') and SEQ ID No. 10 (primer 3') as described in Example No. 2 and the comparison of the sequences obtained with the sequences of the SEQ.ID sequence database.
• n ° 11 to 39 identified the ten amplified strains as: Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus equorum, Staphylococcus schleiferi, Staphylococcus lugdunensis and Staphylococcus gallinarum.
• the decoding of these 20 strains showed 100% agreement between the molecular identification according to the process which is the subject of the present invention and the identification previously established by standard phenotypic methods.
• the other bacteria chosen for being frequently isolated from human or animal clinical samples which may also contain bacteria of the genus Staphylococcus, were not amplified, thus demonstrating the specificity of the primers used for the genus Staphylococcus under the conditions of use for the detection of bacteria of the genus Staphylococcus according to the invention. compared to bacteria of another genus.
• FIG. 1 are represented the PCR amplification products obtained from fifteen coded bacterial strains, comprising 10 strains belonging to the genus Staphylococcus (columns 2 to 5, 8, 9, 11 to 13 and 16) and 5 bacterial strains of genera bacteria other than Staphylococcus (columns 6, 7, 10, 14 and 15).
• Columns 1 and 17 represent the molecular weight marker.
• Columns corresponding to the negative amplification controls (sterile water) and to other strains other than Staphylococcus are not shown.
• the amplification products are obtained after incorporation of the primers SEQ ID No. 7 and SEQ ID No. 10 according to the invention and are visualized by staining with ethidium bromide after electrophoresis on an agarose gel.

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