# Patent

## Title
Molecular identification of Staphylococcus-genus bacteria

## Abstract

The invention relates to a method of detecting, by means of molecular identification, a bacterium from one of the Staphylococcus-type species. The inventive method is characterised in that the following are used: a fragment of the rpoB gene of said bacterium, comprising a nucleotide sequence selected from one of the SEQ. ID. No. 11 to 39 sequences, the reverse sequences and the complementary sequences; or an oligonucleotide comprising a sequence having at least 12 consecutive nucleotide patterns included in one of the SEQ. ID. No. 7 to 10 sequences, in which N represents a nucleotide selected from inosine and an equimolar mixture of 4 different nucleotides selected from A, T, C or G and from the oligonucleotides of the reverse sequences and complementary sequences.

## Background
The present invention pertains to the area of diagnosis. More precisely, the invention concerns a method for the molecular identification of bacteria of the Staphylococcus genus using detection and/or amplifying and sequencing techniques with probes or oligonucleotide primers applied to strains of this bacterial genus.
Bacteria of the Staphylococcus genus are Gram-positive and catalase-positive cocciform bacteria of which 36 species are currently known including 9 with sub-species [Euzé by J P. (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 a few strains of Staphylococcus hyicus [Kloos W E (1995) in Manual of Clinical Micriobiology, pp 282-298, ASM Press]. These species are readily and routinely isolated and cultivated from environmental samples, veterinary clinical samples and human clinical samples [Kloos W E (1986) in Bergey's Manual of Systematic Bacteriology, pp. 1013-1035, Williams & Wilkins]. In man, Staphylococcus aureus is a coagulase-positive species responsible for food poisoning related to the production of an enterotoxin, for staphylococcal toxic shock syndrome and for purulent infections characterized by septic metastases remote from the initial site of infection. Strains of Staphylococcus aureus resistant to methicillin, a first-line antibiotic to fight infection, represent a major problem for public health regarding nosocomial infections, i.e. infections contracted by patients in hospitals and other care institutions. Bacteria belonging to species of the coagulase-negative Staphylococcus genus form part of the normal flora in man. These species are also responsible for nosocomial infections, especially through infection from implanted foreign material, prostheses in particular [Kloos W E (1994) Clin. Microbiol. Rev. 7:117-140].
These different species raise the problem of their identification. Conventional phenotype identification methods are the most frequently used to identify bacteria belonging to species of the Staphylococcus genus [Kloos W E (1991) J. Clin. Microbiol. 29:738-744] and several identification kits and automated units have been developed to assist in the phenotype identification of bacteria of the Staphylococcus genus. However, the extent of identification in routine practice is variable [Grant C E (1994) Diagn. Microbiol. Infect. Dis. 18:1-5; Perl T M (1994) Diagn. Microbiol. Infect. Dis. 18, 151-5; Refshal K (1992) J. Hosp. Infect. 22, 19-31]: for example these systems mostly confuse between bacteria belonging to the Staphylococcus hominis and Staphylococcus warneri species with error rates of 27 to 36% [Gran C E (1994) Diagn. Microbiol. Infect. Dis. 18:1-5; Leven M (1995) J. Clin. Microbiol. 33:1060-3]. Similarly, Staphylococcus schleiferi can be misidentified by automated identification systems [Calvo J. (2000) J. Clin. Microbiol. 38:3887-9]. Molecular methods can in theory give better results when identifying bacteria of the Staphylococcus genus on account of their sensitivity and specificity. The molecular targets currently proposed for the molecular identification of Staphylococcus bacteria comprise the 16S rDNA gene encoding the 16S sub-unit of ribosomal RNA [Bialkowska-Hobrzanska H et al. (1990) Eur. J. Microbiol. Infect. Dis. 9:588-594], the intergenic spacer encoding transfer RNAs [Maes N. et al (1997) J. Clin. Microbiol. 35:2477-2481], the hsp60 gene encoding the heat shock protein 60 [Goh S H et al. (1996) J. Clin. Microbiol. 34:818-823; Goh S H (1997) J. Clin. Microbiol. 35, 3116-3121; Kwok A Y (1999) Int. J. Syst. Bacteriol. 49, 1181-1192] and the femA gene [Vannuffel P et al, Res. Microbiol. 150:129-141]. Hybridization of oligonucleotides is the technique generally offered to target these identification regions. Detection of the nuc gene is limited to bacteria of the Staphylococcus aureus species [Brakstad O G (1992) J. Clin. Microbiol. 30:1654-1660] and a chromosomal fragment has been reported for the identification of bacteria of the Staphylococcus epidermidis species [Martineau F (1996) J. Clin. Microbiol. 34:2888-2893]. There still exists, therefore, a demand for a molecular identification tool for bacteria of the Staphylococcus species which can be routinely used in bacteriology laboratories [Kleeman K T (1993) J. Clin. Microbiol. 31, 1318-1321].
The inventors have shown in this invention that the rpoB gene constitutes a genetic marker enabling the detection and specific identification of the bacteria of each species of the Staphylococcus genus.
More particularly, the present invention concerns sequences of specific nucleic acids of the genus or of each species of the Staphylococcus genus whose nucleotide sequence is drawn from the rpoB gene of said bacteria.
According to Lazcano et al [J. Mol. Evol. (1988) 27:365-376], RNA polymerases are divided into two groups depending upon their origin, one formed by viral RNA- or DNA-dependent RNA polymerases, and the other formed by DNA-dependent RNA polymerases of eukaryotic or prokaryotic origin (archaebacteria and eubacteria). Eubacterial DNA-dependent RNA polymerases are characterized by a simple, multimeric, conserved structure noted “core enzyme” represented by αββ′ or “holoenzyme” represented by αββσ [Yura and Ishihama, Ann. Rev. Genet. (1979) 13:59-97]. Numerous studies have highlighted the functional role, within the multimeric enzyme complex, of the β subunit of eubacterial RNA polymerase. Archaebacterial and eukaryotic RNA polymerases, for their part, have a more complex structure possibly reaching a dozen or even around thirty subunits [Pühlet et al. Proc. Natl. Acad. Sci. USA (1989) 86:4569-4573].
The genes encoding the different αββ′σ subunits of DNA-dependent RNA polymerase in eubacteria, respectively the rpoA, rpoB, rpoC and rpoD genes, are classified in different groups comprising the genes coding for the proteins forming ribosomal subunits or for enzymes involved in the replication and repair of the genome [Yura and Yshihma, Ann. Rev. Genet. (1979) 13:59-97]. Some authors have shown that the sequences of the rpoB and rpoC genes could be used to construct phylogenetic trees ([Rowland et al. Biochem. Soc. Trans. (1992) 21:40 S) enabling separation of the different branches and sub-branches among the kingdoms of the living.
Before setting forth the invention in more detail, different terms used in the description and claims are defined below:
by “nucleic acid extracted from bacteria” is meant either total nucleic acid, or genomic DNA, or messenger RNAs or further DNA obtained from reverse transcription of messenger RNAs;
a “nucleotide fragment” or an oligonucleotide are two synonymous terms denoting a chain of nucleotide patterns characterized by an information sequence of natural (or optionally modified) nucleic acids able to hybridize, like natural nucleic acids, with a complementary or substantially complementary nucleotide fragment under pre-determined conditions of high stringency. The chain may contain nucleotide patterns of different structure from that of natural nucleic acids. A nucleotide fragment (or oligonucleotide) may for example contain up to 100 nucleotide patterns. It generally contains at least 10, and in particular at least 12 nucleotide patterns and may be obtained from a molecule of natural nucleic acid and/or by genetic recombination and/or by chemical synthesis.
a nucleotide pattern is derived from a monomer which may be a natural nucleotide of nucleic acid whose constituent parts are a sugar, a phosphate group and a nitrogenous base chosen from among adenine (A), guanine (G), uracil (U), cytosine (C), thymine (T); or else the monomer is a nucleotide modified in at least one the three preceding constituent parts; by way of example, the modification may occur either at the bases, with modified bases such as inosine which can hybridise with any A, T, U, C or G base, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-dexyuridine or any other modified base able to hybridize, or at the sugar level, for example the replacement of at least one deoxyribose by a polyamide [Nielsen P E et al., Science (1991) 254:1497-15000] or further at the phosphate group level, for example through replacement by esters chosen in particular from among diphosphates, alkylphosphates and phosphorothioates,
by “hybridization” is meant the process during which, under appropriate conditions, two nucleotide fragments having sufficiently complementary sequences are able to associate together through stable, specific hydrogen bonds, to form a double strand. Hybridization conditions are determined by “stringency”, i.e. the strictness of operating conditions. Hybridization is all the more specific the more it is performed under high stringency. Stringency is related in particular to the base composition of a probe/target duplex, and by the extend of mismatch between two nucleic acids. Stringency may also depend upon the parameters of the hybridization reaction, such as concentration and the type of ion species present in the hybridization solution, the type and concentration of denaturing agents and/or hybridization temperature. The stringency of the conditions under which a hybridization reaction is to be performed depends in particular upon the probes used. All this data is well known and the appropriate conditions may possibly be determined in each case through routine experiments. In general, depending upon the length of the probes used, the temperature for hybridization reaction lies between approximately 20 and 65° C., in particular between 35 and 65° C. in a saline solution at a concentration of approximately 0.8 to 1 M.
a “probe” is a nucleotide fragment having hybridization specificity under determined conditions to form a hybridization complex with a nucleic acid having, in this case, a nucleotide sequence included either in a messenger RNA, or in a DNA obtained by reverse transcription of said messenger RNA, the product of transcription; a probe may be used for diagnostic purposes (in particular capture or detection probes) or for therapeutic purposes,
a “capture probe” is a probe that is immobilized or can be immobilized on a solid carrier by any appropriate means, by covalence for example, by adsorption or direct synthesis on a solid. Examples of carriers include microtitration plates and DNA chips,
a “detection probe” is a probe labeled with a marking agent chosen for example from radioactive isotopes, enzymes, in particular enzymes able to act on a chromogenous, fluorigenous or luminescent substrate (in particular a peroxydase or an alkaline phosphatase), chromophor chemical compounds, chromogenous, fluorigenous or luminescent compounds, analogues of nucleotide bases and ligands such as biotin,
a “species probe” is a probe enabling specific identification of the species of a bacterium,
a “genus probe” is a probe enabling specific identification of the genus of a bacterium,
a “primer” is a probe containing for example 10 to 100 nucleotide patterns and having hybridization specificity under determined conditions for enzyme amplification reactions,
by “amplification reaction” is meant an enzyme polymerization reaction, for example in an amplifying technique such as PCR, initiated by primer oligonucleotides and using a DNA polymerase,
by “sequencing reaction” is meant the obtaining of the sequence of a nucleic acid fragment or of a complete gene using an abortive polymerization method with oligonucleotide primers and using said dideoxynucleotides [Sanger F, Coulson A R (1975), J. Mol. Biol. 94: 441] or by multiple hybridizations with multiple probes fixed on a solid carrier such 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 Staphylococcus genus. These four species were chosen by the inventors as representing the four main genetic groups determined on the basis of studies on the 16S gene in bacteria of the Staphylococcus genus, namely the species that are the most divergent phylogenetically among all the species currently described in this genus, so that the alignment of the rpoB sequences obtained in these four species may, most probably, phylogenetically encompass all the rpoB sequences of all the species of this bacterial genus.
The inventors have evidenced the consensus and specific sequences SEQ ID NOs:7 to 10 described in the list of sequences at the end of the description. The inventors have determined said sequences SEQ ID NOs:7 to 10 as being not only consensual between all bacteria of the Staphylococcus genus but also specific to the family of bacteria of the Staphylococcus genus, with the exception of Staphylococcus schleiferi in respect of sequence SEQ ID NO:8.
These sequences are present in the rpoB genes of all bacteria of the Staphylococcus genus and are specific to bacteria of the Staphylococcus genus which may be used as genus probe to detect any bacteria of the Staphylococcus genus with the exception of Staphylococcus schleiferi regarding sequence SEQ ID NO:8.
In sequences SEQ ID NOs:7 and 10, the nucleotide N mentioned in the list of sequences at the end of the description, may represent inosine or an equimolar mixture of 4 different nucleotides chosen from among A, T, C and Gt, or A, U, C and G respectively insofar as, as mentioned in the definitions, an oligonucleotide or a fragment of nucleic acid 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.
When “N” represents said equimolar mixture of nucleotides at a given position, this means that the nucleotide at the said given position indifferently represents A, T, C or G (or respectively A, U, C or G when applicable) and that the oligonucleotide of the invention is more precisely made up of an equimolar mixture of 4 groups of oligonucleotides in each of which N has a different meaning at said given position and respectively represents each of the 4 bases A, T, C or G (or respectively A, U, C or G).
At the position corresponding to a nucleotide N in sequences SEQ ID NOs:7 and 10, variable nucleotides are found in the complementary target sequences in relation to the species of the bacterium under consideration, but all the other nucleotides are conserved in all the species of the bacteria of the Staphylococcus genus. Since “N” represents inosine which is able to hybridize with any base, or an equimolar mixture of the 4 bases A, T, C, G, the sequences SEQ ID NOs:7 and 10 can hybridize with the complementary sequence included in the rpoB gene of all the bacteria of the Staphylococcus genus.
In addition, the consensus sequences SEQ ID NO:9 and SEQ ID NO:10 flank hypervariable sequences whose sequence is specific to each bacterial species of the Staphylococcus genus. The sequences flanked by SEQ ID NOs:9 and 10 may therefore be used a species probes for bacteria of the Staphylococcus genus.
Also, the sequences SEQ ID NOs:9 and 10 were determined as flanking a fragment of the rpoB gene comprising a zone whose variable length is approximately 500 by and forms the shortest specific sequence for each bacterial species of the Staphylococcus genus.
The inventors were therefore able to identify species probes for each of the 29 bacterial species of the Staphylococcus genus studied, corresponding to the sequences SEQ ID NOs:11 to 39 flanked by the consensus sequences SEQ ID NOs:9 and 10.
Consensus sequences SEQ ID NOs:7 to 10 identified in the invention, may be used as amplification or sequencing reaction primer in methods to detect bacteria of the Staphylococcus genus by molecular identification.
Sequences SEQ ID NOs:7 to 10 therefore not only make it possible to prepare genus probes for bacteria of the Staphylococcus genus, but also to detect and identify the species of said bacteria by amplification and sequencing using said sequences as primers.
More precisely, the present invention provides a method for detecting, by identification, a bacterium of a species of the Staphylococcus genus, characterized in that use is made of:
the rpoB gene of said bacterium or a fragment of said rpoB gene of said bacterium, comprising a nucleotide sequence chosen from among 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 nucleotide sequence SEQ ID NO:39, the reverse sequence and the complementary sequence, or
an oligonucleotide comprising a sequence of at least 12 consecutive nucleotide patterns, included in one of sequences SEQ ID NOs:7 to 10, in which N represents a nucleotide chosen from among inosine or an equimolar mixture of 4 different nucleotides chosen from among A, T, C or G, the reverse sequences and the complementary sequences.
Said oligonucleotides preferably comprise 12 to 35 nucleotide patterns, and further preferably said oligonucleotides consist of sequences SEQ ID NOs:7 to 10, the reverse sequences and the complementary sequences.
In one first embodiment of a detection method according to the invention, it is sought to show the presence of a bacterium of the Staphylococcus genus and, in a first variant, the steps are performed in which:
1—at least one genus probe is contacted comprising a said oligonucleotide containing a sequence included in one of sequences SEQ ID NOs:7 to 10, the reverse sequences and the complementary sequences, and
2—the formation or non-formation is determined of a hybridization complex between said genus probe and the nucleic acids of the sample, and the presence is determined of said bacterium of the Staphylococcus genus if there is formation of a hybridization complex.
In a second variant of embodiment of the detection method for a bacterium of the Staphylococcus genus, the steps are performed in which:
1—the amplification primers comprising said oligonucleotides containing a sequence of at least 12 nucleotide patterns included in at least two sequences drawn from sequences SEQ ID NOs:7 to 10, reverse sequences and complementary sequences, are contacted with a sample containing or likely to contain nucleic acids of at least one said bacterium of the Staphylococcus genus, with:
as 5′ primer: an oligonucleotide chosen from among the oligonucleotides comprising a sequence included in one of sequences SEQ ID NOs:7 to 9 or the complementary sequences, preferably an oligonucleotide consisting of said complete sequences, and
as 3′ primer: an oligonucleotide comprising a sequence included in one of sequences SEQ ID NO:10 or 8 or respectively a complementary sequence, preferably an oligonucleotide consisting of said complete sequences.
2—amplification of the nucleic acids is conducted by enzymatic polymerization reaction and the onset or absence of an amplification product is determined, and hence the presence of said bacterium is determined in the sample if an amplification product occurs.
More particularly, in this second variant of the first embodiment, as 5′ primer an oligonucleotide of sequence SEQ ID NO:7 or 9 is used or a complementary sequence, and as 3′ primer an oligonucleotide of sequence SEQ ID NO:10 or respectively a complementary sequence.
In a second embodiment of the method for bacterium detection according to the invention, it is sought to specifically detect a given species of a bacterium of the Staphylococcus genus chosen from among 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 equorum, Staphylococcus epidermis, Staphylococcus cohni, Staphylococcus chromogenes, Staphylococcus carnosus, Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus aureus subs. aureus, Staphylococcus aureus subs. anaerobius, Staphylococcus arlettae, Staphylococcus caprae. 
In a first variant of this second embodiment of the method of the invention, the steps are performed in which:
1—a sample containing or likely to contain nucleic acids of at least one said bacterium is contacted with at least one species probe consisting of a said gene fragment containing 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 NOs:11 to 39, or an oligonucleotide of reverse or complementary sequence, and
2—the formation or absence is determined of a hybridization complex between said probe and the nucleic acids of the sample.
In a second variant of this said second embodiment of the method of the invention in which it is sought to specifically detect a given species of a bacterium of the Staphylococcus genus chosen from among the 29 species cites above, the method comprises the steps in which, in a sample containing or likely to contain nucleic acids of at least one said bacterium:
a) a sequencing reaction is conducted of a fragment of the amplified rpoB gene of said given bacterium using nucleotide primers consisting of oligonucleotides comprising a sequence included in sequences SEQ ID NO:7 or 9 as 5′ primer, and SEQ ID NO:10 as 3′ primer, preferably oligonucleotides consisting of said sequences SEQ ID NO:7 or 9 and SEQ ID NO:10, or their complementary sequences, and
b) the presence or absence is determined of the given species of said bacterium by comparing the sequence of said fragment obtained with the sequence of the complete rpoB gene of said bacterium or the sequence of a fragment of the rpoB gene of said bacterium respectively comprising said sequences SEQ ID NOs:11 to 39 and complementary sequences, and in this way the presence of said bacterium in the sample is determined if the sequence of the fragment obtained is identical to the known sequence of the genus or of the fragment of the rpoB gene of said bacterium.
More particularly, in this second variant:
at step a) the steps are performed comprising:
1—a first amplification of the nucleic acid of said sample with a pair of 5′ and 3′ primers chosen from among oligonucleotides respectively containing sequences SEQ ID NO:7 and respectively SEQ ID NO:10 or their complementary sequences, and the occurrence or absence of an amplification product at step 1 is determined, and
2—a sequencing reaction is conducted of the amplicons determined at step 1 with the 5′ and 3′ primers consisting of oligonucleotides containing sequences SEQ ID NO:9 and respectively SEQ ID NO:10, preferably consisting of said sequences SEQ ID NOs:7 and 10 or their complementary sequences, preferably consisting of said sequences SEQ ID NOs:9 and 10 or their complementary sequences, and
at step b), a comparison is made between the sequences obtained with respectively one of sequences SEQ ID NOs:11 to 39 or their complementary sequences.
A further subject of the present invention is an rpoB gene or gene fragment of a bacterium of the Staphylococcus genus, characterized in that it comprises a sequence such as described in sequences SEQ ID NOs:11 to 29 and 30 to 39.
A further 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 such as described in sequences SEQ ID NOs:3 to 6, as mentioned previously these fragments of rpoB genes and complete genes can be used in particular for a method according to the invention.
The complete sequence of the rpoB gene may be used to identify the bacterium not only by studying its primary sequence, but also by examining the secondary and tertiary structures of the messenger RNA derived from the transcription of the complete DNA sequence.
A further subject of the present invention is a said rpoB gene fragment or oligonucleotide chosen from among the oligonucleotides having a sequence included in sequences SEQ ID NOs:11 to 39 and among the oligonucleotides of reverse sequences and complementary sequences such as defined above.
A further subject of the present invention is an oligonucleotide comprising a sequence of at least 12, preferably 12 to 35 consecutive nucleotide patterns included in one of sequences SEQ ID NOs:7 to 10, in which N represents a nucleotide chosen from among inosine and an equimolar mixture of 4 different nucleotides chosen from among A, T, C or G, and the oligonucleotides of reverse sequences and complementary sequences, preferably consisting of sequences SEQ ID NOs:7 and 10 and the reverse sequences and complementary sequences in which N represents inosine.
Sequences SEQ ID NOs:7 to 39 may be prepared by chemical synthesis using techniques well known to persons skilled in the art, described for example in the article by Itakura K. et al [(1984) Annu. Rev. Biochem. 53:323].
A first application of an oligonucleotide of the invention is its use as probe for the detection, in a biological sample, of bacteria of one of the species of the Staphylococcus genus, which comprises a nucleotide sequence of at least 12 consecutive nucleotide patterns included in one of sequences SEQ ID NOs:7 to 39, and their reverse or complementary sequences.
A probe comprising sequences SEQ ID NOs:7 to 10 will be used as genus probe and a probe comprising one of sequences SEQ ID NOs:11 to 39 will be used as species probe.
The probes of the invention may be used, for diagnostic purposes as mentioned previously, by determining the formation or non-formation of a hybridization complex between the probe and a target nucleic acid in a sample, using all known hybridization techniques and in particular DOT-BLOT techniques [Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor], DNA transfer techniques called SOUTHERN BLOT [Southern E. M., J. Mol. Bio. (1975) 98:503] RNA transfer techniques called NORTHERN BLOT, or so-called “sandwich” techniques, in particular with a capture probe and/or detection probe, said probes being able to hybridize with two different regions of the target nucleic acid, and at least one of said probes (generally the detection probe) being able to hybridize with a region of the target which is specific to the species, the capture probe and the detection probe evidently having nucleotide sequences that are at least partly different.
The nucleic acid to be detected (target) may be DNA or RNA (the first obtained after PCR amplification). For detection of a target of double-strand nucleic acid type, the latter needs to be denatured before implementing the detection method.
The target nucleic acid may be obtained by extraction using known methods for examining nucleic acids in a sample. Denaturing a double strand nucleic acid may be conducted using known chemical, physical or enzymatic denaturing methods, in particular by heating to appropriate temperature, above 80° C.
To implement the above-mentioned hybridization techniques, in particular the “sandwich” techniques, a probe of the invention called a capture probe is immobilized on a solid carrier, and another probe of the invention called a detection probe is labeled with a marking agent. Examples of carriers and marking agents are as defined above.
Advantageously, a species probe is immobilized on a solid carrier, and another species probe is labeled with a marker.
A further application of an oligonucleotide of the invention is its use as nucleotide primer containing a monocatenary oligonucleotide chosen from among the oligonucleotides having a sequence of at least 12 nucleotide patterns included in one of sequences SEQ ID NOs:7 to 39, which can be used in the synthesis of a nucleic acid in the presence of a polymerase using a method known in itself, in particular in amplification methods using such synthesis in the presence of a polymerase (PCR, RT-PCR, etc.). In particular, a primer of the invention may be used for the specific reverse transcription of a messenger RNA sequence of a bacterium belonging to a species of the Staphylococcus genus to obtain a corresponding complementary DNA sequence. Said reverse transcription may form the first stage of the RT-PCR technique, the following stage being PCR amplification of the complementary DNA obtained. It is also possible to use primers of the invention for specific amplification by chain polymerization reaction of the total DNA sequence of the rpoB gene of a species of Staphylococcus genus.
In one particular case, said primer comprising an oligonucleotide of the invention also comprises the sense or anti-sense sequence of a promoter recognized by a RNA polymerase (promoters T7, T3, SP6 for example [Studier F W, B A Moffatt (1986), J. Mol. Biol. 189:113]: said primers can be used in nucleic acid amplification methods involving a transcription step, such as NASBA or 3SR techniques for example [Van Gemen B. et al. Abstract MA 1091, 7th International Conference on AIDS (1991) Florence, Italy].
A further subject of the invention is a nucleotide primer comprising a monocatenary oligonucleotide chosen from among the oligonucleotides having a sequence comprising one of sequences SEQ ID NOs:11 to 29 and 31 to 39, or preferably, consisting of one of sequences SEQ ID NOs:11 to 39 which can be used for total or partial sequencing of the rpoB gene of any strain of a species of the Staphylococcus genus.
Partial or full sequencing of the rpoB gene in any bacterium of the Staphylococcus genus enables the identification of any Staphylococcus bacterium through bio-computer analysis of this sequence and the recognition of new, unknown species of Staphylococcus bacteria.
Preferably, for use as primer or for sequencing rpoB genes, sequences SEQ ID NO: to SEQ ID NO: 0 are used, in which N is the choice inosine, sequences SEQ ID NO:7 and SEQ ID NO:10.
A further subject of the present invention is a diagnosis kit which can be used in a method of the invention, comprising at least one said gene fragment of said oligonucleotide consisting of sequences SEQ ID NOs:7 to 39 and the reverse sequences and complementary sequences, or a said oligonucleotide comprising a sequence included in one of sequences SEQ ID NOs:7 to 10, and/or at least one said rpoB gene fragment of a said bacterium comprising sequences SEQ ID NOs:11 to 39, and the oligonucleotides and gene fragments of reverse sequences and complementary sequences, such as defined above.
In the present description, by “reverse sequences and complementary sequences” is meant the following sequences:
the reverse sequence of said sequence,
the complementary sequence of said sequence, and
the complementary sequence of the reverse sequence of said sequence.
Finally, a last subject of the invention is a gene therapy probe to treat infections caused by a strain belonging to a species of the Staphylococcus genus, said probe comprising an oligonucleotide such as defined above. This gene therapy probe, able to hybridize on the messenger RNA and/or on the genomic DNA of said bacteria, can block phenomena of translation and/or transcription and/or replication.
The principle of gene therapy methods is known and is based especially on the use of a probe corresponding to an antisense strand; the formation of a hybrid between the probe and the sense strand is able to disturb at least one of the decoding steps of genetic information. Gene therapy probes can therefore be used as anti-bacterial medicines, to combat infections caused by bacteria of species of the Staphylococcus genus.
The invention will be better understood with the help of the description given below, divided into examples, which concerns experiments conducted to carry out the invention and which are given solely for illustrative purposes.



FIG. 1 shows visualization of the amplification products obtained in example 3 by ethidium bromide staining after electrophoresis on agarose gel.



EXAMPLE 1
Sequence of the rpoB Gene in Four Species of the Staphylococcus Genus: Staphylococcus saccharolyticus, Staphylococcus lugdunensis, Staphylococcus caprae and Staphylococcus intermedius 
The complete sequence of the ropb gene of bacteria belonging to the species Staphylococcus saccharolyticus, Staphylococcus lugdunensis, Staphylococcus caprae and Staphylococcus intermedius was determined by enzymatic amplification and direct automatic sequencing using consensus primers between the sequences of the rpoB gene in Staphylococcus aureus (GenBank accession NO: X64172) Bacillus subtillis (GenBank accession NO: L43593). This latter bacterial species was chosen as being the Gram-positive species with low guanosine plus cytosine content the closest to species of the Staphylococcus genus (phylogenetic relationship based on comparison of sequences of the 16S rDNA gene).
Several potential consensus primers were investigated to obtain a fragment able to lead to the complete sequence of rpoB genes through successive elongations from a series of specific primers.
These consensus primers have the following sequences:







(SEQ ID NO: 1)







5′-AAA CTT AAT AGA AAT TCA AAC TAA A-3′


 








5′-ATC TGG TAA AGC ATT ACC AA-3′
(SEQ ID NO: 2)






and made it possible to obtain a first fragment F1 having a length of 1 007 base pairs in these four species. From the alignment of the sequence of this first fragment F1 on the sequences of Staphylococcus aureus and Bacillus subtilis, a large number of attempts with theoretically or potentially appropriate primers failed, and finally a succession of oligonucleotide primers was able to be determined to permit amplification and sequencing in successive steps of the entirety of the rpoB gene in the four species Staphylococcus saccharolyticus, Staphylococcus lugdunensis, Staphylococcus caprae and Staphylococcus intermedius. The sequence, the position in relation to the sequence of the rpoB gene of Staphylococcus aureus in GenBank (access number X64172) and the hybridization temperature of these primers (SEQ ID NOs: 40-61, respectively) are given in the following table:

















Temp


Primer
Primer sequence (5′-3′)
Position
(° C.)







 30 F
GGTTTAGGATTAAAAGATGC
30-50
41



(SEQ ID NO: 40)


 


 192 F
GAAGAAGTTGGAGCTACTG
192-211
44



(SEQ ID NO: 41)


 


 806 F
AATAAGAGCAGGGAAAGAAAC
806-827
43



(SEQ ID NO: 42)


 


 920 F
AAAGAAAAGAATGAATGAACTT
920-942
39



(SEQ ID NO: 43)


 


1165 F
TATGCTTATGGTATTTAGCTA
1165-1186
39



(SEQ ID NO: 44)


 


1302 F
AAACTTAATAGAAATTCAAACTAAA
1302-1327
58



(SEQ ID NO: 45)


 


1450 F
GTTCAAACGATAAATAGAGAA
1450-1471
39



(SEQ ID NO: 46)


 


1741 F
GAAACAGATGCTAAAGATGT
1741-1761
41



(SEQ ID NO: 47)


 


1850 F
CCATATACTGCGAGTGGGAA
1850-1870
47



(SEQ ID NO: 48)


 


2245 F
TAGAAATTCAATCAATTAAGTATATG
2245-2271
62



(SEQ ID NO: 49)


 


2309 F
TTGGTAATGCTTTACCAGAT
2309-2329
41



(SEQ ID NO: 50)


 


2334 F
TGCATTACACCAGCAGATATCATTG
2334-2359
70



(SEQ ID NO: 51)


 


2412 F
GATGATATTGACCATTTAGG
2412-243 
41



(SEQ ID NO: 52)


 


2534 F
TGAAAGAATGTCAATTCAAGA
2534-2555
39



(SEQ ID NO: 53)


 


2663 F
AAACCCATTAGCTGAGTT
2663-268 
38



(SEQ ID NO: 54)


 


2995 F
TGGTCGTTTCATGGATGATGAAGTTG
2995-3119
74



(SEQ ID NO: 55)


 


2924 F
AAGATAGCTATGTTGTAGCA
2924-2944
41



(SEQ ID NO: 56)


 


3200 F
CTTAGAGAACGATGACTCTAA
3200-3221
43



(SEQ ID NO: 57)


 


3498 F
TAGTTGGTTTCATGACTTGGGA
3498-3520
46



(SEQ ID NO: 58)


 


3550 F
TTGAAAGTCCAACAAAGCAA
3550-3570
38



(SEQ ID NO: 59)


 


3843 F
GGTAAAGTAACGCCTAAAGGT
3843-3864
45



(SEQ ID NO: 60)


 


4494 F
TGGAGGTATGGGCACTTGAA
4494-4514
47



(SEQ ID NO: 61)









The amplifications were performed under a final volume of 50 μl containing 2.5×102 U Taq polymerase, 1×Taq buffer and 1.8 mM MgCl2, 200 μm dATP, dTTP, dGTP, dCTP and 0.2 μm of each primer. They were performed in accordance with the following program: 35 cycles comprising a denaturing 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 then sequenced using the oligonucleotide sequencing primers (SEQ ID NOs: 62-85, respectively) listed in the following table:
















Temp


Primer
Primer sequence (5′-3′)
Position
(° C.)







1759 R
ACATCTTTAGCATCTGTTTC
1779-1759
48



(SEQ ID NO: 62)


 


1460 R
ATCGTTTGAACGCCACTCTT
1480-1460
45



(SEQ ID NO: 63)


 


1910 R
TCATAGTAAGTTTGCGCCAT
1930-1910
43



(SEQ ID NO: 64)


 


2309 R
ATCTGGTAAAGCATTACCAA
2329-2309
41



(SEQ ID NO: 65)


 


2334 R
CAATGATATCTGCTGGTGTAATGCA
2354-2334
68



(SEQ ID NO: 66)


 


2432 R
CCTAAATGGTCAATATCATC
2452-2432
41



(SEQ ID NO: 67)


 


2573 R
CGAATATTAATTAATTGTTG
2593-2573
34



(SEQ ID NO: 68)


 


2892 R
GTGATAGCATGTGTATCTAAATCA
2912-2892
64



(SEQ ID NO: 69)


 


2915 R
TAACTATCTTCTTCATCAGC
2935-2915
41



(SEQ ID NO: 70)


 


2924 R
TGCTACAACATAGCTATCTT
2944-2924
41



(SEQ ID NO: 71)


 


2995 R
CAACTTCATCATCCATGAAACGACCA
3015-2995
74



(SEQ ID NO: 72)


 


Cm32b
ATGCAACGTCAGGCCGTTCCG
3211-3191
64



(SEQ ID NO: 73)


 


3321 R
AGACGACGAACAGAATTTCA
3341-3321
56



(SEQ ID NO: 74)


 


3610 R
GCTCGAATGATAACGTGATT
3630-3610
43



(SEQ ID NO: 75)


 


4139 R
ACTTGTCCAATGTTCATACG
4159-4139
44



(SEQ ID NO: 76)


 


4502 R
CATATGCTTCAAGTGCCCATA
4523-4502
45



(SEQ ID NO: 77)


 


4508 R
CCAAGTGGTTGTTGTGTAAC
2428-4508
45



(SEQ ID NO: 78)


 


4871 R
TTTAGAGCTTTCACTGTTTG
4891-4871
41



(SEQ ID NO: 79)


 


5000 R
CACCATATGACCAAGAACGAA
5021-5000
45



(SEQ ID NO: 80)


 


5018 R
CAATCAAGGAGCCTACCTCCTT
5040-5018
50



(SEQ ID NO: 81)


 


5030 R
GAAATTATTTACATCAATCAA
5051-5030
36



(SEQ ID NO: 82)


 


5041 R
TAACTATCTTCTTCATCAGC
5061-5041
41



(SEQ ID NO: 83)


 


5085 R
CCCAGTCTTTTGTAGGTCCG
5105-5085
49



(SEQ ID NO: 84)


 


5188 R
CCCATTCTTTCACGACGTAC
5208-5188
47



(SEQ ID NO: 85)









The sequencing reactions were performed using reagents from the ABI Kit: Prism dRhodamine Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems) following the supplier's recommendations and the following program: 30 cycles comprising a denaturing step at 94° C. for 10 sec., a primer hybridization step at 50° C. for 10 sec., and an extension step at 60° C. for 2 minutes. The sequencing products were separated by electrophoresis on polyacrylamide gel using a 377 DNA Sequencer (Perkin) and analyzed to form consensus sequences with Sequence Assembler software (Applied Biosystems). This approach enabled us to determine the complete sequence of the rpoB gene in four species of the Staphylococcus genus:
SEQ ID NO:3: Sequence of the rpoB gene of Staphylococcus saccharolyticus. This sequence measures 3,791 base pairs, has a guanosine plus cytosine content of 36.8% and is deposited with GenBank under Genbank accession number AF325871.








5′ATGAAACTTAATAGAAATTCAAACTAAATCTTAT
(SEQ ID NO: 7)


 


GATTGGTTCCTTAAAGAAGGGTTATTAGAAATGTTT


 


AGAGACATTTCACCAATTGAAGATTTCACAGGCAAC


 


CTATCTTTAGAATTTGTAGATTATAGATTAGGTGAA


 


CCAAATTATGATTTAGAAGAATCTAAAAATCGTGAC


 


GCTACTTATGCTGCACCTCTTCGTGTCAAAGTACGT


 


CTCATTATTAAAGAAACAGGCGAAGTAAAAGAACAA


 


GAAGTCTTCATGGGTGATTTCCCATTAATGACAGAC


 


ACAGGTACATTTGTTATCAATGGTGCTGAGCGTGTT


 


ATCGTGTCTCAATTAGTACGTTCACCATCTGTTTAT


 


TTCAACGAAAAAATTGATAAAAACGGTCGTGAAAAT


 


TATGATGCGACTATTATTCCTAACCGTGGTGCTTGG


 


TTAGAATATGAAACAGATGCTAAAGATGTCGTTTAT


 


GTTCGTATCGATAGAACACGTAAATTACCATTAACT


 


GTATTGTTACGTGCGCTAGGTTTCTCAACTGATCAA


 


GAAATCGTTGATTTAATAGGAGACAGTGAATATTTA


 


CGTAATACATTAGAAAAAGATGGAACTGAAAATACA


 


GAACAAGCTTTATTAGAAATTTATGAACGTTTGCGT


 


CCTGGCGAACCACCAACAGTAGAAAATGCTAAAAGC


 


TTATTATATTCACGTTTCTTCGATCCTAAACGCTAT


 


GATTTAGCAAGTGTAGGTCGTTATAAAGCTAACAAA


 


AAGTTACATTTAAAACACCGTTTATTTAATCAAAAA


 


CTAGCAGAACCAATTGTTAATAGTGAAACAGGTGAG


 


ATTGTAGCGGAAGAAGGTACTGTACTTGATCGTCGT


 


AAACTAGATGAAATCATGGACGTATTGGAGACAAAC


 


GCTAATAGCGAAGTCTTTGAACTTGAAGGTAGTGTC


 


ATTGATGAACCAGTAGAAATTCAATCAATTAAAGTA


 


TATGTTCCTAATGATGAAGAAGGTCGAACTACTACT


 


GTTATTGGTAATGCATTACCAGACTCAGAAGTTAAA


 


TGTATTACTCCGGCTGATATTATCGCCTCAATGAGT


 


TACTTCTTTAACTTATTGAATGGAATTGGTTATACA


 


GATGATATTGACCACTTAGGTAATCGTCGTTTACGT


 


TCAGTTGGTGAATTACTACAAAACCAATTCCGTATC


 


GGTTT


 


GTCTAGAATGGAACGTGTTGTACGTGAGAGAATGTC
(SEQ ID NO: 9)


 


AATTCAAGACACTGATTCTATCACTCCACAACAATT


 


AATTAATATTCGTCCAGTCATTGCATCTATTAAAGA


 


ATTTTTTGGTAGTTCTCAATTATCTCAATTCATGGA


 


CCAAGC


 







(SEQ ID NO: 10)







AAACCCATTAGCTGAGTTGACTCATAAACGTCGTTT


 


ATCAGCTCTAGGACCTGGTGGTTTAACTCGTGAACG


 


CGCTCAAATGGAAGTACGTGACGTGCATTATTCTCA


 


CTACGGTCGTATGTGCCCTATTGAAACACCTGAGGG


 


CCCAAACATTGGATTAATTAACTCATTATCTAGTTA


 


TGCAAGAGTAAATGAATTTGGTTTTATTGAAACACC


 


TTATCGTAAAGTTGATTTAGATACTAATTCAATCAC


 


TGACCAAATTGACTACTTAACTGCTGATGAAGAAGA


 


TAGTTATGTTGTTGCACAAGCAAACTCACGTCTTGA


 


TGAAAATGGGTGCTTCTTAGATGATGAAGTTGTTTG


 


TCGTTTTCGTGGCAATAACACAGTGATGGCTAAAGA


 


AAAAATGGACTATATGGACGTATCACCTAAACAAGT


 


AGTTTCAGCAGCTACTGCATGTATCCCATTCTTAGA


 


AAACGATGACTCAAACCGAGCATTAATGGGTGCAAA


 


CATGCAACGTCAAGCAGTACCATTAATGAACCCAGA


 


AGCGCCATTTGTTGGAACAGGTATGGAACATGTAGC


 








AGCGCGTGACTCAGGTGCAGCAATTACTGCTAAGCA
(SEQ ID NO: 8)


 


TAGAGGACGTGTTGAACATGTTGAGTCTAATGAAGT


 


TTTAGTTCGTCGTTTAGTAGAAGAAAATGGTATTGA


 


ACATGAAGGTGAATTAGATCGCTATCCATTAGCAAA


 


ATTCAAACGTTCAAACTCTGGTACATGTTATAACCA


 


ACGCCCAATTGTTTCTGTTGGAGACGTTGTTGAATA


 


TAACGAAATTTTAGCAGACGGTCCTTCAATGGAACT


 


AGGTGAAATGGCTTTAGGTCGTAACGTAGTTGTAGG


 


TTTCATGACTTGGGACGG


 







(SEQ ID NO: 30)







TTATAACTATGAGGATGCCGTTATCATGAGCGAACG


 


TTTAGTTAAAGATGATGTCTATACATCTATTCATAT


 


CGAAGAATACGAATCAGAAGCACGTGACACTAAATT


 


AGGACCTGAAGAAATTACTCGTGATATTCCTAATGT


 


GTCTGAAAGTGCGCTTAAAAACTTAGACGATCGTGG


 


TATCGTTTATGTTGGTGCCGAAGTTAAAGATGGTGA


 


CATCTTAGTAGGCAAAGTAACGCCTAAAGGTGTAAC


 


GGAACTAACAGCAGAAGAAAGATTATTACATGCTAT


 


TTTCGGTGAAAAGGCTCGTGAAGTTCGTGATACTTC


 


ATTACGTGTACCACATGGTGCAGGGGGCATCGTATT


 


AGATGTAAAAGTCTTCAACCGTGAAGAGGGCGATGA


 


CACTTTATCTCCTGGTGTAAATCAATTAGTACGTGT


 


TTATATCGTTCAAAAACGTAAAATTCATGTAGGGGA


 


TAAAATGTGCGGTCGTCATGGTAATAAAGGTGTTAT


 


TTCTAAAATTGTTCCTGAAGAAGATATGCCATACTT


 


ACCTGATGGTCGACCAATCGACATCATGTTAAATCC


 


ACTTGGTGTACCTTCACGTATGAACATTGGACAAGT


 


GCTAGAATTACACTTAGGTATGGCTGCTAAAAACTT


 


AGGCATCCACATTGCATCACCAGTATTTGATGGTGC


 


TAATGATGATGATGTTTGGTCTACAATCGAAGAGGC


 


CGGCATGGCACGTGATGGTAAGACTGTATTATATGA


 


TGGGCGTACGGGTGAACCGTTTGATAACCGTATTTC


 


TGTAGGTGTAATGTACATGCTTAAACTTGCTCACAT


 


GGTTGATGACAAATTGCATGCACGTTCAACAGGACC


 


ATACTCACTCGTTACACAACAACCACTCGGTGGTAA


 


AGCACAATTTGGTGGACAACGTTTCGGTGAGATGGA


 


GGTATGGGCACTTGAAGCATATGGTGCTGCTTATAC


 


TTTACAAGAAATCTTAACTTATAAATCTGACGATAC


 


AGTAGGACGTGTTAAAACTTACGAATCTATCGTTAA


 


AGGTGAAAACATCTCTAGACCAAGTGTTCCTGAGTC


 


ATTCCGAGTACTGATGAAAGAATTACAAAGTTTAGG


 


ATTAGATGTTAAAGTAATGGATGAGCATGATAATGA


 


AATTGAAATGGCAGATGTTGATGATGAAGATGCAAC


 


GGAACGCAAAGTAGATTTACAACAAAAAAATGCTCC


 


GGAATCACAAAAAGAAACAACTGATTAATAAGCACT


 


TAAGATAAATGAATACTTAAAGGGTATGAAATGATT


 


ATCATTTCAACTTCTTTAGGTATTCGATTTCAATGA


 


AAGTAATCAATCAAATAGCACAGCTAATCTAAATTG


 


AAGGAGGTAGGCTCCTTGATTGATGTAAATAATTTC


 


CATTATATGAAAATAGGATTAGCTTCACCTGAAAAG


 


ATTCGTTCTTGGTCATATGGTGAAGTTAAGAAACCT


 


GAAACAATAAACTATCGTACTTTAAAGCCAGAAAAA


 


GATGGTCTTTTCTGTGAAAGAATTTTCGGACCTACA


 


AAAGACTGGGAAATTTTTAA-3′






SEQ ID NO:4: Sequence of the rpoB gene of Staphylococcus lugdunensis. This sequence measures 3 855 base pairs, has a guanosine plus cytosine content of 36.4% and is deposited with GenBank under Genbank under accession number AF325870.








5′ATGTCTTATGATTGGTTCCTAAAAGAAGGTTTAC
(SEQ ID NO: 7)


 


TAGAAATGTTCCGTGATATCTCACCAATTGAAGATT


 


TCACAGGTAACCTATCATTAGAGTTTGTAGATACAG


 


ATTAGGTGAACCAAAGTATGATTTAGAAGAATCGAA


 


AAATCGTGACGCTACTTATGCTGCACCTCTTCGTGT


 


TAAAGTGCGTCTCGTTATAAAAGAAACAGGTGAAGT


 


TAAAGAGCAAGAAGTATTTATGGGAGACTTCCCATT


 


AATGACAGATACAGGTACGTTTGTTATTAATGGTGC


 


AGAGCGTGTTATTGTATCGCAATTAGTACGTTCACC


 


ATCCGTTTACTTTAATGAAAAAATTGACAAAAACGG


 


ACGAGAAAATTATGATGCTACAATCATTCCTAACCG


 


TGGTGCCTGGTTAGAATACGAAACAGATGCTAAAGA


 


TGTTGTCTATGTTCGTATTGATAGAACTCGTAAATT


 


GCCATTAACTGTCTTATTACGCGCATTAGGCTTTTC


 


AACTGATCAAGAAATTGTTGAGTTGTTAGGCGATAA


 


CGAATACTTGCGTAATACATTAGAAAAAGACGGAAC


 


AGAAAACACTGAACAAGCGTTATTAGAAATTTATGA


 


ACGTTTACGTCCTGGTGAACCACCAACAGTTGAAAA


 


TGCAAAAAGTTTATTATATTCTCGCTTCTTCGATCC


 


GAAACGCTATGATTTAGCAAGCGTTGGACGTTATAA


 


AGCGAACAAAAAATTGCATCTAAAACACCGTTTATT


 


TAATCAAAAATTAGCAGAGCCTATCGTAAACAGCGA


 


AACAGGTGAAATTGTTGCTGAAGAAGGTACTGTATT


 


AGATCGTCGCAAATTAGACGAAATTATGGACGTTCT


 


TGAAACAAATGCGAATAGTGAAGTATTCGAATTAGA


 


AGGAACAGTAATAGACGAACCGGTTGAAATTCAATC


 


AATCAAAGTCTATGTACCAAATGATGAAGAAGGTTG


 


TACAACAACGATAATTGGTAATGCTTTACCAGATTC


 


AGAAGTGAAATGTATCACACCTGCAGATATTATTTC


 


TTCTATGAGTTACTTCTTCAACTTATTAGCTGGCAT


 


TGGTTACACGGATGATATCGATCATTTAGGTAACCG


 


TCGTTTACGTTCAGTTGGTGAGTTATTGCAAAACCA


 


ATTCCGTATTGGTTT


 


ATCAAGAATGGAACGTGTTGTGCGTGAAAGAATGTC
(SEQ ID NO: 9)


 


AATTCAAGATACCGAATCTATCACACCACAACAATT


 


AATTAATATTAGACCAGTTATTGCATCAATTAAAGA


 


ATTCTTTGGTAGTTCTCAATTATCACAATTCATGGA


 


CCAAGC


 







(SEQ ID NO: 10)







TAACCCATTAGCAGAATTAACACACAAACGTCGTTT


 


ATCTGCGTTAGGACCTGGTGGTTTAACACGTGAACG


 


TGCACAAATGGAAGTTCGTGACGTGCATTATTCTCA


 


CTATGGCCGTATGTGTCCGATTGAAACACCAGAGGG


 


TCCAAACATTGGTTTGATTAACTCATTATCTAGTTA


 


TGCGCGTGTCAACGAGTTTGGCTTTATTGAAACGCC


 


TTATCGTAAAGTAGATATTGATACAAATGCAATCAC


 


AGATCAAATTGACTACTTAACTGCTGATGAAGAAGA


 


CAGTTATGTCGTTGCACAAGCGAACTCTCGCCTTGA


 


TGAAAATGGTCGTTTCTTAGATGATGAAGTAGTATG


 


CCGTTTCCGCGGTAATAATACTGTTATGGCTAAAGA


 


AAAAATGGACTACATGGATGTATCTCCTAAACAAGT


 


TGTTTCAGCTGCGACAGCATGTATTCCATTCTTAGA


 


GAACGATGACTCTAACCGTGCATTGATGGGTGCAAA


 


CATGCAACGTCAAGCAGTTCCGTTGATGAACCCTGA


 


AGCGCCGTTCGTAGGAACAGGTATGGAGCATGTTGC


 








TGCTCGTGACTCTGGTGCTGCGATTACTGCAAAATA
(SEQ ID NO: 8)


 


CAGAGGTCGTGTAGAACACGTTGAATCTAATGAAAT


 


CCTAGTGCGTCGATTAATTGAAGAAAATGGAAAAGA


 


ATATGAAGGCGAACTTGATCGCTATCCATTAGCGAA


 


GTTTAAACGCTCTAACTCTGGTACATGTTATAACCA


 


ACGTCCAATTGTTTCTATTGGCGACGTTGTAGAATA


 


CAATGAAATTCTAGCTGACGGTCCATCAATGGAGCT


 


TGGTGAAATGGCATTAGGCCGCAACGTTGTAGTTGG


 


TTTCATGACTTGGGACGG


 


CTATAACTATGAAGATGCTGTCATCATGAGTGAACG
(SEQ ID NO: 4)


 


TTTAGTCAAAGATGACGTTTACACATCTATTCATAT


 


TGAAGAATATGAATCAGAAGCACGTGATACGAAATT


 


AGGACCTGAGGAAATCACACGTGATATTCCTAACGT


 


CTCTGAAAGTGCACTTAAAAACTTAGACGATCGCGG


 


TATTGTTTATGTAGGTGCAGAAGTTAAAGATGGCGA


 


TATTTTAGTAGGTAAAGTAACGCCTAAAGGTGTCAC


 


AGAGCTAACAGCTGAAGAACGTCTATTACATGCAAT


 


CTTTGGTGAAAAAGCACGTGAAGTGCGTGACACTTC


 


ATTGCGTGTACCACATGGTGCTGGCGGTATTGTGCT


 


AGATGTTAAAGTCTTCAACCGTGAAGAAGGAGATGA


 


CACACTTTCTCCAGGTGTTAACCAATTAGTACGCGT


 


ATATATTGTGCAGAAACGTAAAATACACGTTGGGGA


 


CAAAATGTGTGGTCGTCATGGTAACAAAGGTGTCAT


 


TTCTAAGATTGTTCCAGAAGAGGACATGCCTTATTT


 


ACCAGATGGACGTCCAATTGATATTATGTTAAACCC


 


ACTTGGTGTGCCATCACGTATGAACATTGGACAAGT


 


TCTAGAGTTGCATTTAGGTATGGCTGCTAAAAACTT


 


AGGTATTCATGTTGCGTCACCAGTATTTGATGGTGC


 


GAACGATGAAGATGTATGGTCAACAATTGAAGAAGC


 


TGGTATGGCACGTGACGGTAAAACCGTATTATATGA


 


TGGCCGTACAGGTGAGCCATTCGACAACCGTATCTC


 


AGTTGGAGTTATGTACATGCTTAAACTTGCACATAT


 


GGTTGATGACAAATTACATGCTCGTTCAACAGGTCC


 


ATACTCATTAGTTACACAACAACCACTTGGTGGTAA


 


AGCACAATTTGGTGGACAACGTTTCGGTGAGATGGA


 


AGTATGGGCACTTGAAGCTTATGGTGCTGCCTATAC


 


ATTGCAAGAAATCCTTACTTATAAATCTGATGATAC


 


GGTAGGCCGTGTTAAAACATACGAAGCTATCGTTAA


 


AGGTGAAAACATTTCTAGACCAAGTGTTCCTGAATC


 


ATTCCGTGTATTGATGAAAGAACTTCAAAGTTTAGG


 


TTTAGATGTGAAAGTGATGGATGAGCACGATAACGA


 


AATCGAAATGGCAGATGTTGAAGATGAAGATACAAC


 


AGAGCGCAAAGTAGATTTGCAACAAAAAGATGCGCC


 


ACAATCTCAACAAGAAGAAACTGCTGATTAGTCAAT


 


ATATTAGATATAAGGAATGGTGTTAGGAACAAGTGC


 


TACGGATGTTTAAACATAATGTGTTTTGAGTTGCAT


 


CCATCCTAACCTTTCCTTAATTTCAATAGATGTAAA


 


TCAATCAAATGGCACAGCTAATCTAAATTGAAGGAG


 


GTAGGCTCCTTGATTGATGTAAATAATTTCCATTAT


 


ATGAAAATCGGTTTAGCCTCACCTGAAAAAATTCGT


 


TCATGGTCATATGGTGAAGTGAAAAAACCAGAAACA


 


ATTAATTATCGTACGTTAAAACCAGAAAAAGATGGC


 


TTATTCTGTGAGAGAATATTCGGCCCAACTAAAGAT


 


TGGGAATGTAGTTGTGGTAAATACAAACGTGTGCGT


 


TATAAAGGCATGGTTTGTGATAGATGTGGTGTTGT


 


AA-3′






SEQ ID NO:5: Sequence of the rpoB gene of Staphylococcus caprae. This sequence measures 3 698 base pairs, has a guanosine plus cytosine content of 37.4% and is deposited with GenBank under Genbank under accession number AF325868.








5′ATGAAACTTAATAGAAATTCAAACTAAATCTTAC
(SEQ ID NO: 7)


 


GATTGGTCCTTAAAGAAGGTTTATTAGAAATGTTTA


 


GAGACATTTCTCCAATTGAAGATTTCACAGGTAACC


 


TATCTTTAGAATTTGTAGATTATAGATTAGGTGATC


 


CGAAATACGATTTAGAAGAATCTAAAAACCGTGACG


 


CTACTTATGCTGCACCTCTTCGTGTGAAAGTACGTC


 


TCATTATTAAAGAAACAGGCGAAGTGAAGGAACAAG


 


AAGTCTTCATGGGTGATTTCCCATTAATGACTGACA


 


CAGGTACATTCGTTATCAATGGTGCTGAACGTGTTA


 


TCGTTTCTCAATTAGTACGTTCACCATCCGTTTATT


 


TCAACGAGAAAATTGATAAAAATGGACGCGAAAACT


 


ACGATGCAACTATCATTCCTAACCGTGGTGCTTGGT


 


TAGAATATGAAACAGATGCGAAAGATGTAGTATACG


 


TTCGTATCGATAGAACTCGTAAATTACCATTGACAG


 


TATTATTACGTGCACTAGATTTCTCAACTGATCAAG


 


AAATTGTTGATTTACTAGGTGAGAGTGAATATTTAC


 


GTAATACATTAGAAAAAGATGGTACTGAAAATACTG


 


AACAAGCATTATTAGAAATTTATGAACGTTTACGTC


 


CTGGCGAACCACCAACAGTTGAAAATGCTAAAAGCT


 


TATTATACTCACGCTTCTTCGACCCTAAACGTTATG


 


ATTTAGCAAGTGTTGGTCGTTACAAAGCTAACAAAA


 


AGTTACATTTAAAACACCGTTTATTTAATCAAAAAT


 


TAGCAGAACCTATTGTTAATAGTGAAACAGGTGAGA


 


TTGTAGCTGAAGAAGGTACTGTATTAGATCGTCGTA


 


AAATTGACGAAATCATGGACGTTTTAGAAACAAACG


 


CTAACAGTGAAGTTTTCGAATTAGAAGGTAGCGTTA


 


TTGACGAACCTGTTGAAATTCAATCAATTAAAGTCT


 


ATGTACCTAATGATGAAGAAGGTCGCACAACTACTG


 


TAATTGGTAATGCATTACCAGATTCAGAAGTTAAAT


 


GTATTACTCCAGCTGATATCATTGCGTCAATGAGTT


 


ATTTCTTCAACTTATTAAATGGTATTGGTTATACAG


 


ATGATATCGACCACTTAGGTAACCGTCGTTTACGTT


 


CAGTTGGTGAACTTTTACAGAACCAATTCCGTATCG


 


GTTT


 


ATCAAGAATGGAACGTGTTGTTCGTGAAAGAATGTC
(SEQ ID NO: 9)


 


TATTCAAGACACTGATTCAATCACACCACAACAATT


 


AATCAACATTCGTCCGGTTATTGCGTCTATTAAAGA


 


ATTCTTCGGAAGTTCACAATTATCGCAATTCATGGA


 


CCAAGC


 







(SEQ ID NO: 10)







TAACCCATTAGCTGAGTTGACTCATAAACGTCGTCT


 


ATCAGCATTAGGACCTGGTGGTTTAACGCGTGAACG


 


TGCCCAAATGGAAGTGCGTGACGTTCACTATTCTCA


 


CTATGGCCGTATGTGTCCAATCGAAACACCTGAGGG


 


ACCAAACATTGGTTTAATCAACTCATTATCAAGTTA


 


TGCACGAGTAAATGAATTTGGTTTTATTGAAACACC


 


TTATCGTAAAGTAGATTTAGATACGAATTCTATCAC


 


TGACCAAATTGATTACTTAACTGCTGATGAAGAAGA


 


TAGTTATGTTGTTGCCCAAGCGAACTCTCGTTTAGA


 


CGAAAATGGTCGTTTCTTAGATGACGAAGTTGTTTG


 


TCGTTTCCGTGGTAATAACACAGTTATGGCTAAAGA


 


GAAAATGGACTACATGGATGTATCTCCTAAACAAGT


 


AGTATCTGCAGCGACAGCTTGTATTCCATTCTTAGA


 


AAATGATGACTCTAACCGTGCATTAATGGGTGCGAA


 


CATGCAACGTCAAGCAGTACCATTGATGAATCCAGA


 


AGCGCCATTTGTTGGTACAGGTATGGAACATGTAGC


 








CGCACGTGATTCAGGTGCAGCGATTACTGCTAAACA
(SEQ ID NO: 8)


 


TAGAGGACGCGTTGAACACGTTGAATCTAACGAAGT


 


ATTAGTACGTCGTTTAGTAGAAGAAAACGGCACTGA


 


ACATGAAGGTGAATTAGATCGTTACCCATTAGCTAA


 


ATTCAAACGTTCAAACTCTGGTACATGTTATAACCA


 


ACGTCCAATGTTTCTGTTGGTGATGTAGTAGAATAC


 


AATGAAATTTTAGCTGACGGTCCTTCAATGGAATTA


 


AGGTTGAAATGGCATAGGGACGTAACGTTGTTAGTT


 


GGTTTCATGACTTGGGACGG


 


TTATAACTACGAGGATGCTGTTATCATGAGTGAACG
(SEQ ID NO: 5)


 


TTTAGTTAAAGATGACGTTTATACTTCTATTCACAT


 


TGAAGAATATGAATCTGAAGCTCGTGATACTAAGTT


 


AGGACCTGAAGAAATTACTCGTGACATTCCTAACGT


 


ATCTGAAAGTGCACTTAAAAACTTAGACGATCGCGG


 


TATCGTTTATGTTGGTGCTGAAGTTAAAGACGGTGA


 


CATCTTAGTAGGTAAAGTAACGCCTAAAGGTGTAAC


 


TGAATTAACAGCTGAAGAAAGATTATTACATGCTAT


 


CTTCGGTGAAAAGGCTCGTGAAGTCCGCGATACATC


 


ATTACGTGTACCACATGGTGCAGGCGGTATCGTTCT


 


AGATGTTAAAGTATTCAATCGTGAAGAAGGCGATGA


 


TACGTTATCTCCAGGTGTAAACCAATTGGTACGTGT


 


TTATATCGTTCAAAAACGTAAAATTCATGTAGGGGA


 


CAAAATGTGTGGTCGTCACGGTAACAAAGGTGTTAT


 


CTCTAAAATTGTTCCTGAAGAAGATATGCCATACTT


 


ACCAGATGGTCGTCCAATCGACATCATGTTAAACCC


 


ACTTGGTGTACCATCACGTATGAACATCGGACAAGT


 


ACTTGAGTTGCATTTAGGTATGGCTGCTAAGAACTT


 


AGGCATCCATGTAGCATCTCCAGTATTCGATGGTGC


 


AAACGATGATGATGTATGGTCAACAATTGAAGAAGC


 


AGGTATGGCTCGTGATGGTAAAACTGTATTATACGA


 


TGGACGTACAGGTGAACCATTCGATAACCGTATTTC


 


TGTAGGTGTCATGTACATGCTTAAACTTGCTCACAT


 


GGTTGACGATAAATTACACGCACGTTCAACTGGACC


 


ATACTCACTTGTTACACAACAACCACTTGGTGGTAA


 


AGCACAATTCGGTGGTCAACGCTTCGGTGAGATGGA


 


GGTATGGGCACTTGAAGCATATGGTGCTGCATACAC


 


ATTACAAGAAATCTTAACTTATAAATCTGACGATAC


 


AGTAGGTCGTGTTAAAACTTACGAATCTATCGTTAA


 


AGGTGAAAATATCTCTAGACCAAGTGTTCCAGAATC


 


ATTCAGAGTATTGATGAAAGAATTACAAAGTTTAGG


 


ATTAGATGTTAAAGTGATGGACGAGCAAGACAACGA


 


AATTGAAATGGCGGACGTTGATGATGAAGATGCAAC


 


TGAACGCAAAGTAGATTTACAACAAAAAAATGCTCC


 


CGAATCACAAAAAGAAACAACTGATTAATAAGCACT


 


TAAGATAAATGAATCCTAAAGAGGTTATGAGATGGT


 


TGCCATTTCAACCTCTTTAAGGTATTCGATTTCAAT


 


GAATGTAAATCAATCAAATAGCACAGCTAATCTAAA


 


TTGAAGGAGGTAGGCTCCTTGATTGATGTAAATAAT


 


TTCCATTATATGAAAATAGGATTAGCTTCACCTGAA


 


AAAATTCGTTCTTGGTCTTATGGTGAAGTTAA-3′






SEQ ID NO:6: Sequence of the rpoB gene of Staphylococcus intermedius. This sequence measures 3 851 base pairs, has a guanosine plus cytosine content of 39.2% and is deposited with GenBank under Genbank accession number AF325869.








5′ATGTAAACTTAATAGAAATTCMAACTAAATCGTA
(SEQ ID NO: 7)


 


TGATTGGTTCTTAAAAGAAGGTTTATTAGAAATGTT


 


CCGTGATATTTCTCCTATTGAAGACTTCACGGGTAA


 


TCTTTCATTAGAATTTGTTGATTATAGATTAGGTGA


 


ACCAAAGTATGATTTAGAAGAATCAAAAAACCGTGA


 


TGCAACATACGCGGCACCATTACGTGTGAAAGTTCG


 


TTTAATCATTAAAGAAACAGGCGAAGTGAAAGATCA


 


AGAAGTATTTATGGGTGATTTCCCATTAATGACAGA


 


AACAGGTACTTTTGTGATTAACGGGGCAGAACGTGT


 


TATCGTATCACAATTAGTCCGTTCACCATCTGTATA


 


CTTCAATGAAAAATTAGATAAAAACGGATGCGTGAA


 


TTATGATGCGACAGTCATTCCTAACCGTGGTGCTTG


 


GTTGGAATATGAAACAGATGCGAAAGATGTCGTTTA


 


TGTGCGTATCGATAGAACGAGAAAGTTACCATTAAC


 


AGTATTATTACGTGCGTTAGGTTATTCAACAGACCA


 


AGAAATTATTGAATTAATTGGGGATAATGAATATTT


 


ACGTAATACATTAGAAAAAGATAGCACAGAAAATAC


 


AGAGCAAGCATTACTTGAAATTTATGAACGTTTACG


 


TCCAGGTGAACCACCTACTGTAGAAAACGCAAAAAG


 


CTTATTATACTCACGTTTCTTTGACCCTAAACGTTA


 


TGATTTAGCAAGCGTTGGACGTTATAAAGCAAACAA


 


AAAGTTACATTTAAAACACCGCCTATTCAATCAAAA


 


ATTAGCTGAACCGATCGTTAATACTGAAACAGGCGA


 


AATTGTTGCTGAAGAAGGCACTGTTTTAGATCGTCG


 


TAAATTAGATGAAATTATGGACGTTCTTGAAACAAA


 


TGCGAATGCACAAGTTTATGAACATTCCAAACGGAT


 


CATTGATGAGCCAGTAGAAATTCAATCAATTAAAGT


 


ATATGTACCGAATGATGATGAAGAACGTACAACAAC


 


AGTTATTGGTAATGCATTCCCAGATTCAGAAGTGAA


 


ATGTATTACACCGGCTGATATTGTGGCATCTATGTC


 


ATACTTCTTCAACCTATTACATGGTATTGGTTACAC


 


AGACGATATTGACCACCTTGGTAACCGCCGTCTACG


 


TTCAGTTGGTGAGTTGTTACAAAACCAATTCCGTAT


 


CGGTTT


 


ATCAAGAATGGAACGTGTGGTACGTGAAAGAATGTC
(SEQ ID NO: 9)


 


TATTCAAGATACAGACTCTATCACACCGCAACAATT


 


AATTAATATTCGTCCAGTGATTGCATCAATTAAAGA


 


GTTCTTTGGTAGCTCGCAATTATCTCAATTCATGGA


 


CCAAGC


 







(SEQ ID NO: 10)







GAACCCACTTGCTGAGTTGACTCACAAACGTCGTCT


 


ATCAGCATTAGGACCTGGTGGTTTAACGCGTGAACG


 


TGCTCAAATGGAAGTGCGTGACGTACACTACTCTCA


 


CTATGGTCGTATGTGTCCAATCGAAACACCTGAGGG


 


ACCAAACATTGGTTTGATCAACTCATTATCTAGTTA


 


TGCACGTGTGAACGAATTTGGTTTTATCGAAACACC


 


ATATCGTAAAGTTGATATTGAAACAAATACGATTAC


 


TGACCAAATCGACTACTTAACTGCTGATGAAGAAGA


 


TAGTTATGTTGTCGCACAAGCGAACTCACGTCTTGA


 


TGAAAACGGTCGCTTTATTGATGATGAGATTGTATG


 


TCGTTTCCGTGGTAACAACACAACGATGGCGAAAGA


 


AAAAATGGACTACATGGACGTATCGCCGAAACAAGT


 


TGTATCAGCTGCGACAGCGTGTATCCCATTCTTAGA


 


AAACGATGACTCTAACCGTGCGTTAATGGGTGCGAA


 


CATGCAGCGTCAAGCGGTACCGTTGTTAAACCCTGA


 


ATCTCCATTTGTAGGTACAGGTATGGAACACGTTGC


 








TGCACGTGACTCAGGTGCTGCTGTCATTTCTAAATA
(SEQ ID NO: 8)


 


TCGCGGTCGTGTTGAACATGTCCAATCTAGCGAGAT


 


TTTAGTCCGTCGTTTAGTTGAAGAAAACGGTCAAGA


 


AGTAGATGGTACGTTAGATCGTTATCCATTAGCGAA


 


ATTTAAACGTTCGAACTCAGGTACATGTTATAACCA


 


ACGTCCAATCATCGCAAAAGGTGACATTGTGGAAAA


 


AGGCGAAATCCTTGCTGATGGTCCTTCAATGGAACT


 


TGGTGAAATGGCATTAGGTCAGAAACGTAGTAGTTG


 


GTTCATGACTTGGGACGG


 


TTATAACTATGAGGATGCCGTTATCATGAGTGAACG
(SEQ ID NO: 6)


 


TTTGGTTAAAGATGATGTGTACACGTCTATTCATAT


 


TGAAGAATACGAATCAGAAGCGCGTGACACAAAACT


 


TGGACCTGAAGAAATCACACGTGATATTCCTAACGT


 


ATCTGAAAATGCACTGAAAAACTTAGATGATCGCGG


 


TATCGTTTATGTAGGTGCGGAAGTTAAAGACGGCGA


 


CATCTTAGTGGGTAAAGTAACGCCAAAAGGTGTAAC


 


AGAATTAACTGCAGAAGAACGTTTATTACATGCAAT


 


CTTTGGTGAAAAAGCACGTGAAGTACGTGATACATC


 


ATTACGTGTACCTCACGGCGCGGGCGGTATTGTACT


 


TGATGTTAAAGTGTTCAATCGTGAAGAAGGCGATGA


 


TTCACTTTCACCAGGTGTGAACCAACTCGTACGTGT


 


TTACATTGTTCAAAAACGTAAAATTCATGTAGGGGA


 


CAAAATGTGTGGTCGTCACGGTAACAAAGGTGTCAT


 


CTCTAAAATTGTTCCTGAAGAAGACATGCCGTACTT


 


ACCAGACGGTCGTCCAATCGACATCATGTTGAACCC


 


ACTCGGTGTACCATCTCGTATGAACATCGGACAAGT


 


TTTAGAGCTCCACTTAGGTATGGCAGCTAAAAACTT


 


AGGTATCCACGTTGCATCACCAGTATTCGATGGTGC


 


GAACGATGATGACGTATGGTCTACAATTGAAGAAGC


 


AGGTATGGCACGTGATGGTAAAACTGTCCTTTACGA


 


TGGACGTACAGGTGAACCATTCGACAACCGTATCTC


 


TGTAGGTGTCATGTACATGCTGAAACTTGCACACAT


 


GGTTGATGACAAGCTTCACGCACGTTCTACAGGACC


 


TTACTCACTTGTTACACAACAACCGCTTGGTGGTAA


 


AGCACAGTTTGGTGGACAAAGATTTGGTGAGATGGA


 


GGTATGGGCACTTGAAGCATACGGTGCAGCATACAC


 


ATTACAAGAAATCCTCACATACAAATCAGATGACAC


 


AGTAGGTCGTGTGAAAACTTACGAAGCTATCGTTAA


 


AGGTGAAAACATCTCAAGACCAAGTGTTCCTGAATC


 


ATTCCGCGTATTGATGAAAGAATTACAAAGTTTAGG


 


TCTTGACGTTAAAGTGATGGACGAACAAGATAACGA


 


AATTGAAATGCGTGACTTAGACGATGATGATATTCC


 


AGATCGCAAAGTCAACATTCAACCATCAACTGTTCC


 


TGAATCACAAAAAGAATTTAACGAATAATGATGAAT


 


TGTAGATAAGATTAAACGGAATAGAAACACTTGGTT


 


AAGCTTGAGTTTGTGTTCAAATGTGACAGTTGAAAT


 


ACAACAGATGTCATGTACGATTAATCTATTCGGAAA


 


TGTGATCGGAATCCAACGAGAGGGCTTGGGTTTCGA


 


TGCATATCCGATACTGCAACATTTTTAAGATAAATT


 


GTAAATCAATCAACTAGCACAGTTAATTTAAACTAA


 


AGGAGGTAGGCTCCTTGATTGATGTAAATAAATTCC


 


ATTACATGAAAATAGGACTCGCTTCACCTGAAAAAA


 


TTCGTTCTTGGTCATATGGTGAGGTCAAAAAGCCAG


 


AAACAATTAACTACCGTACGTTAAAACCAGAAAAAG


 


ATGGTAA-3′






This sequence measures 3 852 base pairs, has a guanosine plus cytosine content of 39.2% and is deposited with Genbank under accession no AF325869.
EXAMPLE 2
Partial Sequencing of the rpoB Gene of 26 Species of the Staphylococcus Genus
The alignment of the rpoB sequence determined in bacteria of the species Staphylococcus aureus, Staphylococcus lugdunensis (GenBank accession AF325870), Staphylococcus intermedius (GenBank accession AF325869), Staphylococcus saccharolyticus (GenBank accession AF325871) and Staphylococcus caprae (GenBank accession AF325868) permitted the determination of the consensus sequences of the following oligonucleotides respectively positioned at position 2491-2511 and 3554-357 of the rpoB gene in Staphylococcus aureus: 
SEQ ID NO:7: 5′-AACCAATTCCGTATNGGTTT-3′ (where N represents inosine).
SEQ ID NO:8: 5′-CCGTCCCAAGTCATGAAAC-3′ theoretically determining the amplification of a fragment of 1 063 base pairs in all species of the Staphylococcus genus.
SEQ ID NO:8 is used as 3′ primer and therefore represents the complementary reverse sequence of the direct strand represented in sequences SEQ ID NOs:3 to 6 at position 3554-3573 in Staphylococcus aureus. 
The inventors have determined the position of these two primers SEQ ID NO:7 and SEQ ID NO:8 paying heed to the following criteria:
1. sequence flanked by these two primers specific to the species of the bacterium. This condition is verified after alignment of 1063 bp fragments with all the sequences of the rpoB bacterial genes available in computer data banks.
2—search for the shortest possible identification region so as to increase the sensitivity of molecular detection as much as possible,
3—search for a region close to the one previously worked by inventors in the area of enterobacteria [Mollet C. (1997) Mol. Microbiol., 26:1005-11] so as to tend towards a working area common to these two bacterial genus and family.
4—primer length of 18 to 22 bp,
5—primer sequences having close melting points
6—primer sequence not permitting self-hybridization or complementarity.
In silico analysis predicted that these two oligonucleotides SEQ ID NO:7 and SEQ ID NO:8 should enable PCR amplification of a fragment of 1 063 base pairs of the rpoB gene in all species of the Staphylococcus genus. In reality, the primer of sequence SEQ ID NO:8 did not adhere to a rare species for undetermined reasons. Laboratory experiments showed that the species of the genus: Staphylococcus schleiferi was not amplified by this pair of oligonucleotide primers, demonstrating the uncertain nature of predictions made on primers. 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 SEQ ID NO:7 oligonucleotide in a PCR amplification reaction, effectively enabled the obtaining of an amplicon of the rpoB gene having a size of 771 base pairs (size for the reference species Staphylococcus aureus) in 29 species of the Staphylococcus genus tested by the inventors:
SEQ ID NO:10: 5′GCIACITGITCCATACCTGT-3′ (where n=inosine)
SEQ ID NO:10 is used as 3′ primer. This is why it corresponds to the complementary reverse sequence of the sequences of the direct strand represented on sequences SEQ ID NOs:3 to 6.
This amplification product is then sequenced by incorporating two sequencing primers, SEQ ID NO:9 (located at position 2643-2660 of the rpoB gene in bacteria of the species Staphylococcus aureus) and SEQ ID NO:10.

    
    
        SEQ ID NO:9: 5′-CAA TTC ATG GAC CAA GC-3′.
    
    


This last primer was determined to pay heed to the constraints of a sequencing primer, i.e. a size of more than 15 mothers, not hybridizing with the second primer used for sequencing, and flanking a zone of approximately 500 base pairs in general whose sequence is specific to each species in the Staphylococcus genus.
By using this second set of oligonucleotides of sequences SEQ ID NO:9/SEQ ID NO:10, the inventors were therefore finally able to determine the partial sequence of the rpoB gene in 29 species of the Staphylococcus genus listed below (SEQ ID NO:11 to SEQ ID NO:39).
The fragment of the rpoB gene was amplified with the PCR technique using 35 amplification cycles each comprising a denaturing phase at 94° C. for 10 seconds, a hybridization phase of primers SEQ ID NOs:7 and 8 or SEQ ID NOs:7 and 10 at 52° C. for 20 seconds, and an elongation phase at 72° C. for 60 seconds. The amplification product was visualized after ethidium bromide staining.
The bacteria representing these 29 species of the Staphylococcus genus are the following:












Species
GenBank accession n°
Reference







Staphylococcus caprae
AF325868 (SEQ ID NO: 39)
CIP 104000T


Staphylococcus 
AF325890 (SEQ ID NO: 27)
CIP 103504T


gallinarum




Staphylococcus aureus
AF325894 (SEQ ID NO: 37)
CIP 103780T


subsp. anaerobius




Staphylococcus aureus
X64172 (SEQ ID NO: 36)
CIP 103428T


subsp. aureus




Staphylococcus
AF325872 (SEQ ID NO: 30)
CIP 81.55T


epidermidis




Staphylococcus
AF325888 (SEQ ID NO: 26)
CIP 81.56T


haemolyticus




Staphylococcus
AF325869 (SEQ ID NO: 23)
CIP 81.60T


intermedius




Staphylococcus
AF325870 (SEQ ID NO: 20)
CIP 103642T


lugdunensis




Staphylococcus
AF325871 (SEQ ID NO: 17)
CIP 103275T


saccharolyticus




Staphylococcus schleiferi
AF325886 (SEQ ID NO: 15)
CIP 103643T


subsp. schleiferi




Staphylococcus xylosus
AF325883 (SEQ ID NO: 11)
CIP 81.66T


Staphylococcus capitis
AF325885 (SEQ ID NO: 34)
ATCC 27840T


subsp. capitis




Staphylococcus arlettae
AF325874 (SEQ ID NO: 38)
ATCC 43957T


Staphylococcus warneri
AF325887 (SEQ ID NO: 12)
ATCC 27836T


Staphylococcus hominis
AF325875 (SEQ ID NO: 25)
ATCC 27844T


Staphylococcus simulans
AF325877 (SEQ ID NO: 13)
ATCC 27848T


Staphylococcus
AF325873 (SEQ ID NO: 16)
ATCC 15305T


saprophyticus




Staphylococcus equorum
AF325882 (SEQ ID NO: 29)
ATCC 43958T


Staphylococcus cohnii
AF325893 (SEQ ID NO: 31)
ATCC 29974T


subsp. Cohnii




Staphylococcus
AF325889 (SEQ ID NO: 35)
ATCC 33753T


auricularis




Staphylococcus carnosus
AF325880 (SEQ ID NO: 33)
ATCC 51365T


subsp. Carnosus




Staphylococcus kloosii
AF325891 (SEQ ID NO: 22)
ATCC 43959T


Staphylococcus
AF325892 (SEQ ID NO: 32)
ATCC 43764T


chromogenes




Staphylococcus hyicus
AF325876 (SEQ ID NO: 24)
ATCC 11249T


subsp. hyicus




Staphylococcus pulveris
AF325879 (SEQ ID NO: 18)
CCUG 33938T


Staphylococcus muscae
AF325884 (SEQ ID NO: 19)
CIP 1035641T


Staphylococcus lentus
AF036973 (SEQ ID NO: 21)
ATCC 49574


Staphylococcus felis
AF325878 (SEQ ID NO: 28)
CIP 103366T


Staphylococcus sciuri
AF325881 (SEQ ID NO: 14)
ATCC 29062T





ATCC: American Tissue Culture Collection;


CIP: Collection de l'Institut Pasteur;


Ttype strain.






The fragments of, in general, approximately 500 base pairs of the rpoB gene of the bacteria of species belonging to the Staphylococcus genus whose sequence is specific to each species of this genus and therefore enabling molecular identification of the bacteria of the 29 species tested are:
SEQ ID NO:11: Partial sequence of the rpoB gene in Staphylococcus xylosus, measuring 518 base pairs:







(SEQ ID NO: 11)







5′TTCAGGGTTCATCAATGGCACTGCTTGACGTTGCATGTTTGCACCCAT


 


CAATGCACGGTTAGAGTCATCATTTTCTAAGAAAGGAATACATGCTGTCG


 


CAGCAGAAACAACTTGTTTTGGTGAAACGTCCATGTAATCCATTTTTTCT


 


TTAGCCATAACTGTGTTATTACCACGGAAACGACAAACAACTTCATCATC


 


TAAGAAACGACCATTTTCATCTAATTTAGAGTTGGCTTGTGCTACCACAT


 


AACTATCCTCTTCATCAGCTGTTAAGTAATCGATTTGCTCAGTAATGCTG


 


TTTGTTTCAAGGTCTACTTTACGATAAGGTGTTTCAATGAAACCAAATTC


 


ATTCACACGTGCATAACTAGACAATGAGTTGATAAGTCCAATGTTTGGAC


 


CTTCAGGCGTTTCGATTGGACACATACGGCCATAGTGAGAATAGTGAACG


 


TCACGTACTTCCATTTGAGCACGTTCACGTGTTAAACCACCAGGTCCTAG


 


AGCAGATAAACGACGTTTGT-3′






SEQ ID NO:12: Partial sequence of the rpoB gene in Staphylococcus warneri, measuring 507 base pairs:







(SEQ ID NO: 12)







5′TTCAGGATTCATCAATGGTACTGCTTGACGTTGCATGTTCGCACCCAT


 


TAATGCACGGTTAGAGTCATCGTTTTCTAAGAATGGAATACAAGCTGTAG


 


CGGCTGAAACAACCTGCTTAGGTGAAACGTCCATGTAATCCATTTTTTCT


 


TTAGCCATTACTGTGTTATTACCACGGAAACGACAAACTACTTCGTCATC


 


TATGAAACGTCCGTTTTCATCTAAACGTGAATTCGCTTGGGCAACAACAT


 


AACTATCTTCTTCGTCAGCAGTTAAATAATCAATTTGGTCTGTAATCGCA


 


TTAGTGTCTAAATCCACTTTACGATATGGTGTTTCAATGAAACCAAATTC


 


GTTTACACGTGCATAACTAGATAATGAGTTGATTAATCCAATGTTTGGAC


 


CCTCTGGCGTTTCAATTGGACACATACGACCATAGTGAGAATAGTGTACG


 


TCACGTACCTCCATTTGTGCACGTTCACGTGTTAAACCACCAGGTCCTAA


 


AGCAGATAA-3′






SEQ ID NO:13: Partial sequence of the rpoB gene in Staphylococcus simulans, measuring 518 base pairs:









(SEQ ID NO: 13)







5′TTCAGGGTTCATCAATGGTACTGCTTGACGTTGCATGTTCGCACCCAT


 


TAACGCACGGTTAGAGTCATCGTTTTCTAAGAATGGGATACATGCTGTCG


 


CTGCAGATACAACTTGTTTAGGAGAAACGTCCATATAGTCCATTTTCTCT


 


CTATCCATAGTTGTGTTGTTACCACGGAAACGACAAACGATTTCTTCGTC


 


TAAGAAACGACCTTCGTCATCTAAACGTGAGTTCGCTTGCGCAACAACAT


 


AGCTGTCTTCTTCGTCTGCAGTAAGGTAATCGATTTGATCTGTTACCGCA


 


TTTTTCTCATGGTCAACTTTACGATATGGTGTTTCAATGAAACCAAATTC


 


ATTAACACGCGCATAACTTGATAATGAGTTGATTAAACCGATGTTCGGAC


 


CCTCTGGTGTCTCGATTGGACACATACGGCCATAGTGAGAGTAATGCACG


 


TCACGTACTTCCATTTGTGCACGTTCACGTGTTAAACCACCAGGTCCAAG


 


TGCAGATAGACGACGTTTAT-3′






SEQ ID NO:14: Partial sequence of the rpoB gene in Staphylococcus sciuri, measuring 507 base pairs:









(SEQ ID NO: 14)







5′TTCTGGGTTCATTAAAGGTACCGCTTGACGTTGCATGTTTGCACCCAT


 


AAGCGCACGGTTAGAGTCATCGTTTTCTAAGAATGGAATACATGCTGTCG


 


CTGCAGAAACAACTTGTTTAGGAGATACATCCATGTAGTCCATGCGTTCT


 


TTAGGTTTAGTAGTGTTGTCCCCACGGAAACGACAAAGAACTTCATCATC


 


AACGAATTTACCTGTTTCATCAAGTACAGAGTTTGCTTGTGCAACTACAT


 


AGCTGTCTTCTTCGTCAGCTGTTAAGTAGTCGATTCTGTCAGTAACTTGG


 


TTTGTCTCGATGTTTACCTTACGATAAGGTGTTTCAATGAAACCAAATTC


 


ATTAACTCTTGCATAACTTGATAATGAGTTGATTAAACCAATGTTTGGTC


 


CCTCAGGCGTTTCAATTGGACACATACGACCATAGTGAGAGTAGTGAACG


 


TCACGTACTTCCATACCAGCACGCTCACGAGTTAAACCACCCGGTCCTAA


 


TGCTGATAG-3′






SEQ ID NO:15: Partial sequence of the rpoB gene in Staphylococcus schleiferi, measuring 518 base pairs:









(SEQ ID NO: 15)







5′TTCTGGGTTTAACAATGGTACTGCTTGACGTTGCATGTTCGCACCCAT


 


CAATGCACGGTTAGAGTCATCGTTTTCTAAAAACGGAATACATGCTGTCG


 


CAGCTGAAACAACTTGTTTAGGCGATACGTCCATGTAGTCCATTTTTTCT


 


TTAGCCATAGTTGTGTTGTTACCACGGAAACGACAAACGATTTCGTCATC


 


GATAAAACGTCCGTTTTCATCAAGTCTTGAGTTCGCTTGGGCAACAACAT


 


AACTGTCTTCTTCATCAGCAGTAAGGTAATCAATACGGTCTGTAATTGTG


 


TTTGTTTCAAGGTCTACTTTTCTGTATGGAGTTTCAATGAAACCAAATTC


 


ATTCACACGTGCATAACTTGAAAGTGAGTTGATCAAACCAATGTTTGGAC


 


CCTCTGGTGTCTCGATTGGACACATACGGCCATAGTGAGAATAGTGTACG


 


TCACGAACTTCCATTTGTGCACGTTCACGTGTTAAACCACCAGGCCCTAA


 


AGCTGATAAACGACGTTTGT-3′






SEQ ID NO:16: Partial sequence of the rpoB gene in Staphylococcus saprophyticus, measuring 518 base pairs:









(SEQ ID NO: 16)







5′TTCTGGATTCATCAATGGCACTGCTTGACGTTGCATGTTCGCACCCAT


 


CAATGCACGGTTAGAGTCATCGTTTTCTAAGAAAGGAATACATGCTGTCG


 


CTGCAGAAACAACTTGTTTAGGTGAGACATCCATATAATCCATTTTTTCT


 


TTGGCCATAACTGTATTATTACCACGGAAACGACAAACAACTTCGTCTGC


 


TATGAAACGGCCATTTTCGTCTAATGTTGAGTTTGCTTGTGCTACAACAT


 


AGCTATCTTCTTCATCAGCTGTTAAATAGTCAATTTGATCCGTGATTGAA


 


TTCGTTTCAAGATCCACTTTACGGTAAGGTGTTTCAATAAAGCCGAATTC


 


ATTTACACGCGCATAACTAGATAACGAGTTAATAAGTCCGATGTTTGGAC


 


CCTCTGGCGTTTCAATTGGACACATACGGCCATAGTGAGAATAGTGAACG


 


TCACGTACTTCCATTTGAGCACGTTCACGCGTTAAACCACCAGGTCCTAG


 


AGCTGATAAACGACGTTTAT-3′






SEQ ID NO:17: Partial sequence of the rpoB gene in Staphylococcus saccharolyticus, measuring 556 base pairs:









(SEQ ID NO: 17)







5′AAACCCATTAGCTGAGTTGACTCATAAACGTCGTTTATCAGCTCTAGG


 


ACCTGGTGGTTTAACTCGTGAACGCGCTCAAATGGAAGTACGTGACGTGC


 


ATTATTCTCACTACGGTCGTATGTGCCCTATTGAAACACCTGAGGGCCCA


 


AACATTGGATTAATTAACTCATTATCTAGTTATGCAAGAGTAAATGAATT


 


TGGTTTTATTGAAACACCTTATCGTAAAGTTGATTTAGATACTAATTCAA


 


TCACTGACCAAATTGACTACTTAACTGCTGATGAAGAAGATAGTTATGTT


 


GTTGCACAAGCAAACTCACGTCTTGATGAAAATGGGTGCTTCTTAGATGA


 


TGAAGTTGTTTGTCGTTTTCGTGGCAATAACACAGTGATGGCTAAAGAAA


 


AAATGGACTATATGGACGTATCACCTAAACAAGTAGTTTCAGCAGCTACT


 


GCATGTATCCCATTCTTAGAAAACGATGACTCAAACCGAGCATTAATGGG


 


TGCAAACATGCAACGTCAAGCAGTACCATTAATGAACCCAGAAGCGCCAT


 


TTGTTGGA-3′






SEQ ID NO:18: Partial sequence of the rpoB gene in Staphylococcus pulveris, measuring 508 base pairs:









(SEQ ID NO: 18)







5′TTCAGGATTCATTAAAGGCACTGCTTGACGTTGCATGTTTGCACCCAT


 


AAGCGCACGGTTAGAGTCATCGTTTTCTAAGAAAGGAATACATGCTGTCG


 


CAGCAGAAACAACCTGTTTAGGTGATACATCCATGTAATCCATACGTTCT


 


TTAGGTTTCGTAGTATTATCCCCACGGAAACGACAAAGTACTTCATCATC


 


AACGAATTTACCTGTTTCATCAAGTACTGAGTTTGCTTGCGCTACAACAT


 


AGCTGTCTTCTTCGTCAGCTGTTAAATAGTCAATTCTGTCAGTAACTTGG


 


TTTGTTTCGATATTAACCTTACGATAAGGCGTTTCAATAAAACCAAATTC


 


ATTAACTCTCGCATAACTTGATAAAGAGTTAATTAAACCGATGTTTGGTC


 


CCTCAGGTGTTTCAATTGGACACATACGACCATAGTGAGAATAGTGAACG


 


TCACGTACTTCCATACCAGCACGTTCACGAAGTTAAACCGCCGGGTCCTA


 


ATGCTGATAG-3′






SEQ ID NO:19: Partial sequence of the rpoB gene in Staphylococcus muscae, measuring 518 base pairs:









(SEQ ID NO: 19)







5′TTCAGGATTCAACAATGGCACCGCTTGACGTTGCATGTTCGCACCCAT


 


TAAGGCACGGTTAGAGTCATCGTTTTCTAAGAATGGAATACATGCTGTCG


 


CAGCAGAAACAACTTGCTTCGGCGATACGTCCATGTAGTCCATTTTCTCT


 


TTTGCCATTGTTGTGTTGTTACCACGGAAACGACATACAATCTCATCATC


 


AATAAAGCGACCATTTTCATCTAAACGTGAGTTCGCTTGTGCAACCACAT


 


AACTATCTTCTTCATCAGCAGTTAAATAGTCGATTTGATCAGTGATTGTG


 


TTCGTCTCGATATCAACTTTACGATATGGTGTTTCAATGAAACCAAATTC


 


ATTAACACGTGCATAACTAGATAGTGAGTTGATCAAACCAATGTTCAGTC


 


CCTCTGGTGTCTCAATCGGACACATACGACCATAGTGAGAGTAGTGAACG


 


TCACGCACTTCCATTTGTGCACGTTCACGTGTCAAACCACCAGGCCCTAA


 


TGCTGAAAGACGACGCTTAT-3′






SEQ ID NO:20: Partial sequence of the rpoB gene in Staphylococcus lugdunensis, measuring 556 base pairs:









(SEQ ID NO: 20)







5′TAACCCATTAGCAGAATTAACACACAAACGTCGTTTATCTGCGTTAGG


 


ACCTGGTGGTTTAACACGTGAACGTGCACAAATGGAAGTTCGTGACGTGC


 


ATTATTCTCACTATGGCCGTATGTGTCCGATTGAAACACCAGAGGGTCCA


 


AACATTGGTTTGATTAACTCATTATCTAGTTATGCGCGTGTCAACGAGTT


 


TGGCTTTATTGAAACGCCTTATCGTAAAGTAGATATTGATACAAATGCAA


 


TCACAGATCAAATTGACTACTTAACTGCTGATGAAGAAGACAGTTATGTC


 


GTTGCACAAGCGAACTCTCGCCTTGATGAAAATGGTCGTTTCTTAGATGA


 


TGAAGTAGTATGCCGTTTCCGCGGTAATAATACTGTTATGGCTAAAGAAA


 


AAATGGACTACATGGATGTATCTCCTAAACAAGTTGTTTCAGCTGCGACA


 


GCATGTATTCCATTCTTAGAGAACGATGACTCTAACCGTGCATTGATGGG


 


TGCAAACATGCAACGTCAAGCAGTTCCGTTGATGAACCCTGAAGCGCCGT


 


TCGTAGGA-3′






SEQ ID NO:21: Partial sequence of the rpoB gene in Staphylococcus lentus, measuring 507 base pairs:









(SEQ ID NO: 21)







5′TTCAGGGTTCATTAAAGGTACTGCTTGACGTTGCATGTTCGCACCCAT


 


TAAGGCACGGTTAGAGTCATCGTTTTCAAGGAAAGGAATACATGCTGATG


 


GTGCAGAAACAACTTGTTTAGGAGATACATCCATGTAATCCATACGTTCT


 


TTAGGTTTAGTAGTGTTGTCACCACGGAAACGACAAAGAACTTCATCGTC


 


GACGAATCTACCAGTTTCATCTAATACTGAGTTTGCTTGTGCAACAACAT


 


AACTATCTTCTTCATCAGCAGTTAGATAATCAATTCTGTCTGTTACTTGG


 


TTAGTTTCGATATTAACTTTACGATATGGTGTTTCAATAAAGCCAAACTC


 


GTTAACTCTAGCATAACTTGAAAGTGAGTTGATTAAACCAATGTTTGGTC


 


CCTCTGGTGTCTCAATCGGACACATACGACCATAGTGAGAATAGTGAACG


 


TCACGTACTTCCATACCAGCACGTTCACGAGTTAAACCGCCGGGTCCAAG


 


CGCTGATAG-3′






SEQ ID NO:22: Partial sequence of the rpoB gene in Staphylococcus kloosii, measuring 505 base pairs:









(SEQ ID NO: 22)







5′TTCACGGTTCATCAATGGTACCGCTTGACGTTGCATGTTCGCACCCAT


 


TAAGGCACGGTTAGAGTCATCGTTTTCTAAGAAAGGAATACATGCTGTCG


 


CAGCCGAAACAACTTGTTTTGGTGATACGTCCATGTAGTCCATTTTTTCT


 


TTCGCCATAACTGTGTTGTTACCACGGAAACGACAAACTACTTCATCATC


 


TAAGAAACGACCATTTTCATCTAATTTAGAGTTAGCTTGCGCTACCACAT


 


AGCTATCTTCTTCATCAGCTGTTAAATAGTCAATTTGATCTGTGATTGAA


 


TTAGTTTCTAAATCAACTTTACGGTATGGTGTTTCGATAAAGCCAAATTC


 


ATTAACACGTGCATAACTTGATAATGAGTTGATAAGTCCAATGTTTGGAC


 


CCTCTGGCGTTTCGATTGGACACATACGACCATAGTGAGAATAGTAACGT


 


CACGCACTTCCATTTGAGCACGTTCACGAGTTAAACCACCAGGTCCAAGC


 


CAGATAG-3′






SEQ ID NO:23: Partial sequence of the rpoB gene in Staphylococcus intermedius, measuring 556 base pairs:









(SEQ ID NO: 23)







5′GAACCCACTTGCTGAGTTGACTCACAAACGTCGTCTATCAGCATTAGG


 


ACCTGGTGGTTTAACGCGTGAACGTGCTCAAATGGAAGTGCGTGACGTAC


 


ACTACTCTCACTATGGTCGTATGTGTCCAATCGAAACACCTGAGGGACCA


 


AACATTGGTTTGATCAACTCATTATCTAGTTATGCACGTGTGAACGAATT


 


TGGTTTTATCGAAACACCATATCGTAAAGTTGATATTGAAACAAATACGA


 


TTACTGACCAAATCGACTACTTAACTGCTGATGAAGAAGATAGTTATGTT


 


GTCGCACAAGCGAACTCACGTCTTGATGAAAACGGTCGCTTTATTGATGA


 


TGAGATTGTATGTCGTTTCCGTGGTAACAACACAACGATGGCGAAAGAAA


 


AAATGGACTACATGGACGTATCGCCGAAACAAGTTGTATCAGCTGCGACA


 


GCGTGTATCCCATTCTTAGAAAACGATGACTCTAACCGTGCGTTAATGGG


 


TGCGAACATGCAGCGTCAAGCGGTACCGTTGTTAAACCCTGAATCTCCAT


 


TTGTAGGT-3′






SEQ ID NO:24: Partial sequence of the rpoB gene in Staphylococcus hyicus, measuring 518 base pairs:









(SEQ ID NO: 24)







5′CTCTGGGTTCAATAAAGGCACGGCTTGACGTTGCATGTTCGCACCCAT


 


TAATGCACGGTTCGAGTCATCGTTTTCTAAGAATGGGATACATGCTGTCG


 


CCGCAGAAACAACTTGTTTCGGTGATACGTCCATGTAATCCATTTTTTCT


 


TTAGCCATTGTTGTATTGTTCCCACGGAAACGACAAACGATTTCGTCGTC


 


GATAAAGCGTCCATTTTCATCTAAACGTGAGTTCGCTTGGGCAACAACAT


 


AACTGTCTTCTTCATCCGCAGTTAAGTAATCAATTTGATCTGTTATTGTA


 


TTCGTTTCAAGGTCCACTTTACGGTAAGGCGTTTCAATGAAACCAAATTC


 


GTTAACACGCGCATAACTTGAAAGTGAGTTGATTAATCCAATGTTTGGAC


 


CCTCTGGCGTTTCGATTGGACACATACGACCGTAGTGAGAGTAGTGAACG


 


TCACGCACTTCCATTTGGGCACGTTCACGCGTTAAACCACCAGGTCCTAA


 


TGCAGATAAACGACGTTTGG-3′






SEQ ID NO:25: Partial sequence of the rpoB gene in Staphylococcus hominis, measuring 518 base pairs:









(SEQ ID NO: 25)







5′TTCAGGATTCATCAATGGTACTGCTTGACGTTGCATGTTCGCACCCAT


 


TAACGCACGGTTAGAGTCATCGTTTTCAAGGAATGGAATACAAGCTGTCG


 


CTGCTGATACTACTTGTTTAGGAGATACATCCATGTAGTCCATTTTTTCT


 


TTTGCCATAACAGTGTTGTTACCACGGAAACGACATACCACTTCATCATC


 


TAGGAAACGACCATTTTCATCTAAACGAGAATTGGCTTGTGCAACTACAT


 


AGCTATCTTCTTCATCAGCAGTTAAATAATCAATTTGATCAGTAATCGAA


 


TTGGTATCAATATCTACTTTACGATATGGTGTTTCGATAAAACCAAATTC


 


ATTTACACGTGCATAACTAGATAATGAGTTAATTAAACCAATGTTTGGTC


 


CCTCTGGTGTTTCAATTGGACACATACGACCATAGTGAGAATAGTGTACG


 


TCACGAACTTCCATTTGTGCACGTTCACGTGTTAAACCACCAGGTCCTAA


 


AGCAGAAAGACGACGTTTAG-3′






SEQ ID NO:26: Partial sequence of the rpoB gene in Staphylococcus haemolyticus, measuring 507 base pairs:









(SEQ ID NO: 26)







5′TTCTGTGTTCATCAATGGTACTGCtTGACGTTGCATGTTTGCACCCAT


 


TAATGCACGGTTAGAGTCATCATTTTCAAGGAAAGGAATACATGCTGTCG


 


CAGCTGAAACTACTTGTTTAGGAGATACGTCCATGTAGTCCATTTTCTCT


 


TTAGCCATAACTGTGTTATTACCACGGAAACGACATACGACTTCATCATC


 


TAAGAAACGACCATTTTCATCTAAGCGAGAGTTCGCTTGGGCAACTACAT


 


AGCTATCTTCTTCATCAGCAGTTAAGTAGTCGATTTGATCTGTAATAGAG


 


TTAGTGTCTAAGTCTACTTTACGATATGGTGTTTCAATGAAACCAAATTC


 


ATTCACACGTGCATAACTTGATAATGAGTTAATCAAACCAATGTTTGGTC


 


CCTCTGGAGTCTCGATCGGACACATACGACCATAGTGAGAGTAGTGAACG


 


TCACGTACTTCCATTTGAGCACGTTCACGTGTTAAACCACCAGGTCCTAA


 


TGCAGAAAG-3′






SEQ ID NO:27: Partial sequence of the rpoB gene in Staphylococcus gallinarum, measuring 507 base pairs:









(SEQ ID NO: 27)







5′TTCAGGATTCATCAAAGGTACAGCTTGACGTTGCATGTTCGCACCCAT


 


CAATGCACGGTTAGAGTCATCGTTTTCTAAGAAAGGAATACATGCTGTCG


 


CAGCAGATACAACCTGTTTAGGTGATACATCCATGTAGTCCATTTTTTCT


 


TTTGCCATTACAGTGTTGTTACCACGGAAACGACAAACGACTTCATCTTC


 


TACGAAACGACCATTTTCATCTAATACAGAGTTTGCTTGTGCTACTACAT


 


AACTGTCTTCTTCATCAGCTGTTAAGTAGTCAATTTGATCTGTAATAGAT


 


TGTGTTTCAATATCAACTTTACGATATGGTGTTTCAATGAAACCAAATTC


 


ATTTACACGCGCATAACTTGATAATGAGTTGATAAGTCCGATGTTTGGAC


 


CCTCAGGTGTTTCGATTGGACACATACGGCCATAGTGAGAATAGTGAACG


 


TCACGTACTTCCATTTGAGCACGTTCACGAGTTAAACCACCAGGTCCTAA


 


TGCTGATAG-3′






SEQ ID NO:28: Partial sequence of the rpoB gene in Staphylococcus felis, measuring 518 base pairs:









(SEQ ID NO: 28)







5′TTCGGGATTCATTAAAGGTACAGCTTGACGTTGCATGTTCGCACCCAT


 


TAATGCACGGTTAGAGTCATCGTTTTCTAAGAATGGGATACATGCCGTCG


 


CAGCAGAAACGACTTGCTTAGGCGATACGTCCATGTAGTCCATTTTTTCT


 


TTGGCCATCGTTGTATTGTTTCCGCGGAAACGACATACAATCTCGTCATC


 


CAAGAAACGGCCTTCTTCGTCTAATCGTGCGTTTGCTTGTGCAACAACAT


 


AACTATCTTCTTCATCAGCTGTAAGATAGTCAATTTGGTCTGTAATTTTA


 


TTTGTCTCAAGATCGACTTTACGATATGGTGTTTCGATAAATCCAAATTC


 


GTTAACACGTGCATAACTTGATAATGAGTTGATTAATCCGATGTTCGGCC


 


CCTCTGGCGTTTCAATAGGACACATGCGACCATAGTGAGAGTAGTGAACG


 


TCACGCACTTCCATCTGTGCACGTTCTCTCGTTAAACCACCAGGTCCTAA


 


TGCGGATAGACGACGTTTAT-3′






SEQ ID NO:29: Partial sequence of the rpoB gene in Staphylococcus equorum, measuring 507 base pairs:









(SEQ ID NO: 29)







5′TTCAGGATTCATCAATGGCACTGCTTGACGTTGCATGTTTGCACCCAT


 


CAATGCACGGTTAGAGTCATCGTTTTCTAAGAAAGGAATACATGCTGTCG


 


CAGCAGAAACAACTTGTTTAGGTGAAACATCCATGTAGTCCATTTTTTCT


 


TTAGCCATAACTGTGTTATTACCACGGAAACGACAAACAACTTCGTCTTC


 


TACGAAACGACCATTTTCATCTAATACAGAGTTTGCTTGAGCTACTACAT


 


AGCTGTCTTCTTCGTCAGCTGTTAAGTAGTCAATTTGGTCTGTGATTGAA


 


TGTGTTTCAAGATCTACTTTACGGTAAGGTGTTTCAATGAAACCAAATTC


 


ATTCACACGCGCATAACTAGATAGTGAGTTGATAAGTCCGATATTCGGAC


 


CCTCTGGTGTTTCGATTGGACACATACGACCATAGTGAGAATAGTGAACG


 


TCACGTACTTCCATTTGAGCACGTTCACGTGTTAAACCGCCGGGTCCTAA


 


TGCTGATAA-3′






SEQ ID NO:30: Partial sequence of the rpoB gene in Staphylococcus epidermidis, measuring 518 base pairs:









(SEQ ID NO: 30)







5′TTCAGGATTCATTAAAGGCACCGCTTGACGTTGCATGTTTGCTCCCAT


 


TAACGCACGGTTAGAGTCGTCATTTTCTAAGAATGGAATACATGCTGTTG


 


CTGCTGAAACAACTTGTTTTGGTGATACGTCCATGTAATCCATTTTTTCT


 


TTAGCCATAACAGTGTTATTACCACGGAAACGACAAACAACTTCATCATC


 


TAAGAAACGACCATTTTCATCAAGTCTAGAATTAGCCTGTGCAACAACGT


 


AGCTATCCTCTTCATCAGCTGTCAAATAATCTATTTGATCAGTGATTGAG


 


TTTGTATCTAAATCCACTTTACGATATGGCGTTTCAATAAAACCAAATTC


 


ATTCACTCTAGCATAACTTGACAATGAGTTTATTAAACCAATATTAGGAC


 


CCTCAGGTGTTTCAATTGGACACATACGCCCATAGTGAGAGTAGTGAACG


 


TCACGCACTTCCATTTGAGCACGTTCACGTGTTAATCCACCAGGCCCTAG


 


AGCAGATAAACGACGTTTGT-3′






SEQ ID NO:31: Partial sequence of the rpoB gene in Staphylococcus cohnii, measuring 507 base pairs:









(SEQ ID NO: 31)







5′TTCTGGATTCATCAATGGGACTGCTTGACGTTGCATGTTCGCACCCAT


 


TAATGCACGGTTAGAGTCATCGTTTTCTAAGAATGGAATACATGCTGTTG


 


CTGCAGAAACAACCTGTTTAGGAGATACATCCATGTAATCCATTTTTTCT


 


TTTGCCATAACTGTGTTATTACCACGGAAACGACAAACAACTTCATCATC


 


TAAGAAGCGACCATTTTCATCTAACTTAGAATTTGCTTGTGCTACTACAT


 


AGCTATCTTCTTCGTCAGCTGTTAAATAATCAATTTGATCTGTGATACTA


 


TTCGTTTCAAGATCTACTTTACGATATGGCGTTTCAATGAAACCAAATTC


 


ATTTACACGTGCATAACTTGATAATGAGTTAATCAAACCAATGTTTGGTC


 


CCTCTGGTGTTTCGATTGGACACATACGACCGTAGTGAGAGTAGTGAACG


 


TCACGCACTTCCATTTGAGCACGTTCACGTGTTAAACCACCAGGTCCTAA


 


TGCTGATAG-3′






SEQ ID NO:32: Partial sequence of the rpoB gene in Staphylococcus chromogenes, measuring 507 base pairs:









(SEQ ID NO: 32)







5′CTCAGGATTTAACAAAGGCACCGCTTGACGTTGGATGTTCGCACCCAT


 


TAACGCACGGTTAGAGTCATCGTTTTCTAAGAACGGAATACATGCAGTTG


 


CCGCAGAAACAACTTGCTTCGGTGATACGTCCATGTAATCCATTTTTTCT


 


TTAGCCATTGTTGTATTGTTCCCACGGAAACGACAAACGATTTCGTCGTC


 


GATAAAGCGTCCATTTTCATCTAAACGTGAGTTCGCTTGGGCAACAACAT


 


AACTGTCTTCTTCGTCCGCAGTTAAATAATCAATTTGATCAGTAATTGCG


 


TTCGTTTCAAGGTCTACTTTACGATACGGCGTTTCAATAAAACCAAATTC


 


ATTAACACGCGCATAACTTGAAAGTGAGTTGATTAATCCAATATTTGGAC


 


CCTCTGGTGTTTCGATTGGACACATACGACCGTAGTGAGAATAGTGAACG


 


TCACGCACTTCCATTTGAGCACGTTCACGTGTTAAACCACCTGGTCCTAA


 


AGCAGATAA-3′






SEQ ID NO:33: Partial sequence of the rpoB gene in Staphylococcus carnosus, measuring 1,025 base pairs:









(SEQ ID NO: 33)







5′TTCTGGATTCATCAATGGTACCGCTTGACGTTGCATGTTCGCACCCAT


 


TAATGCACGGTTAGAGTCATCGTTTTCTAAGAATGGGATACAAGCTGTCG


 


CAGCTGATACTACTTGTTTTGGTGATACGTCCATGTAGTCCATTTTGTCT


 


CTGTCCATCATTGTGTTGTTACCACGGAAACGACAAACAACTTCTTCGCT


 


GATGAAGTGACCTTCATCATCTAAACGAGAGTTCGCTTGGGCTACAACAT


 


AGCTGTCTTCTTCGTCAGCTGTTAGATAGTCGATTTGATCAGTTACAGTA


 


TTAGTTTCAAGGTCAACTTTACGGTATGGTGTTTCAATAAAACCGAACTC


 


GTTAACACGTGCATAACTTGATAATGAGTTGATCAAACCAATGTTTGGAC


 


CCTCAGGAGTTTCGATTGGACACATACGGCCATAGTGAGAATAGTGAACG


 


TCACGTACTTCCATTTGAGCACGTTCACGAGTTAAACCACCAGGTCCTAA


 


TGCAGATAATTCTGGATTCATCAATGGTACTGCTTGACGTTGCATGTTCG


 


CACCCATTAATGCACGGTTAGAGTCATCATTTTCTAAGAATGGAATACAA


 


GCTGTCGCTGCAGATACTACTTGTTTAGGAGATACATCCATGTAGTCCAT


 


TTTCTCTTTAGCCATAACTGTGTTATTACCACGGAAACGACAAACAACTT


 


CGTCATCTAAGAAACGACCATTTTCGTCTAAACGAGAGTTCGCTTGGGCA


 


ACAACATAACTATCTTCTTCATCAGCAGTTAAGTAATCAATTTGGTCAGT


 


GATAGAATTCGTATCTAAATCTACTTTACGATAAGGTGTTTCAATAAAAC


 


CAAATTCATTTACTCGTGCATAACTTGATAATGAGTTGATTAAACCAATG


 


TTTGGTCCCTCAGGTGTTTCGATTGGACACATACGGCCATAGTGAGAATA


 


GTGAACGTCACGCACTTCCATTTGGGCACGTTCACGCGTTAAACCACCAG


 


GTCCTAATGCTGATAGACGACGTTTAT-3′






SEQ ID NO:34: Partial sequence of the rpoB gene in Staphylococcus capitis, measuring 518 base pairs:









(SEQ ID NO: 34)







5′TTCAGTGTTCATCAATGGTACCGCTTGACGTTGCATGTTCGCACCCAT


 


TAATGCACGGTTAGAGTCATCGTTTTCTAAGAATGGAATACATGCTGTAG


 


CTGCTGATACAACTTGTTTAGGTGATACGTCCATGTAATCCATTTTTTCT


 


TTTGCCATAACTGTGTTATTACCACGGAAACGACAAACAACCTCGTCATC


 


TAAGAAACGACCATTTTCGTCTAAACGTGAGTTGGCTTGGGCAACTACAT


 


AGCTATCTTCTTCATCAGCAGTTAAGTAATCGATTTGATCTGTGATAGAG


 


TTCGTATCTAAATCAACTTTACGATACGGTGTCTCGATGAAACCAAATTC


 


ATTTACTCGCGCATAACTTGATAATGAGTTAATTAAACCAATATTTGGAC


 


CCTCTGGTGTTTCAATTGGACACATACGACCATAGTGTGAGTAATGAACG


 


TCACGTACTTCCATTTGAGCACGTTCACGAGTTAAACCACCAGGTCCTAA


 


TGCTGATAGACGACGTTTTG-3′






SEQ ID NO:35: Partial sequence of the rpoB gene in Staphylococcus auricularis, measuring 507 base pairs:









(SEQ ID NO: 35)







5′TTCTGGGTTCATTAAAGGTACCGCTTGACGTTGCATGTTTGCACCCAT


 


AAGCGCACGGTTAGAGTCATCGTTTTCTAAGAATGGAATACATGCTGTCG


 


CTGCAGAAACAACTTGTTTAGGAGATACATCCATGTAGTCCATGCGTTCT


 


TTAGGTTTAGTAGTGTTGTCCCCACGGAAACGACAAAGAACTTCATCATC


 


AACGAATTTACCTGTTTCATCAAGTACAGAGTTTGCTTGTGCAACTACAT


 


AGCTGTCTTCTTCGTCAGCTGTTAAGTAGTCGATTCTGTCAGTAACTTGG


 


TTTGTCTCGATGTTTACCTTACGATAAGGTGTTTCAATGAAACCAAATTC


 


ATTAACTCTTGCATAACTTGATAATGAGTTGATTAAACCAATGTTTGGTC


 


CCTCAGGCGTTTCAATTGGACACATACGACCATAGTGAGAGTAGTGAACG


 


TCACGTACTTCCATACCAGCACGCTCACGAGTTAAACCACCCGGTCCTAA


 


TGCTGATAG-3′






SEQ ID NO:36: Partial sequence of the rpoB gene in Staphylococcus aureus, measuring 518 base pairs:









SEQ ID NO: 36)







5′TTCTGGATTCATCAAAGGCACTGCTTGACGTTGCATGTTCGCACCCAT


 


CAATGCACGGTTTGAGTCATCATTTTCTAAGAATGGAATACATGCTGTCG


 


CTGCTGAAACAACTTGCTTCGGCGATACATCCATATAATCCATTTTTTCT


 


TTAGCCATAACTGTGTTGTTACCACGGAAACGACATACAACTTCATCATC


 


CATGAAACGACCATTTTCATCTAATTTAGAGTTTGCTTGTGCTACAACAT


 


AGCTATCTTCTTCGTCAGCTGTTAAATAGTCAATTTGATCAGTGATAGCA


 


TGTGTATCTAAATCAACTTTACGATATGGTGTTTCAATAAAGCCGAATTC


 


ATTTACACGTGCATAACTTGATAATGAGTTAATCAATCCAATGTTTGGTC


 


CCTCAGGTGTTTCAATTGGACACATACGGCCATAGTGAGAGTAGTGAACG


 


TCACGTACTTCCATTTGAGCACGTTCACGTGTTAAACCACCAGGTCCTAA


 


TGCTGATAGACGACGTTTAT-3′






SEQ ID NO:37: Partial sequence of the rpoB gene in Staphylococcus aureus anaerobius, measuring 507 base pairs:









(SEQ ID NO: 37)







5′TTCTGGATTCATCAAAGGCACTGCTTGACGTTGCATGTTCGCACCCAT


 


CAATGCACGGTTTGAGTCATCATTTTCTAAGAATGGAATACATGCTGTCG


 


CTGCTGAAACAACTTGCTTCGGCGATACATCCATATAATCCATTTTTTCT


 


TTAGCCATAACTGTATTGTTACCACGGAAACGACATACAACTTCATCATC


 


CATGAAACGACCATTTTCATCTAATTTAGAGTTTGCTTGTGCTACAACAT


 


AGCTATCTTCTTCGTCAGCTGTTAAATAGTCAATTTGATCAGTGATAGCA


 


TGTGTATCTAAATCAACTTTACGATATGGTGTTTCAATAAAGCCGAATTC


 


ATTTACACGTGCATAACTTGATAATGAGTTAATCAATCCAATGTTTGGTC


 


CCTCAGGTGTTTCAATTGGACACATACGGCCATAGTGAGAGTAGTGAACG


 


TCACGTACTTCCATTTGAGCACGTTCACGTGTTAAACCACCAGGTCCTAA


 


TGCCGATAG-3′






SEQ ID NO:38: Partial sequence of the rpoB gene in Staphylococcus arlettae, measuring 518 base pairs:









(SEQ ID NO: 38)







5′TTCACGGTTCATCAACGGTACTGCTTGACGTTGCATGTTCGCACCCAT


 


TAATGCACGGTTAGAGTCATCGTTTTCTAAGAAAGGAATACATGCCGTTG


 


CAGCTGAAACTACTTGCTTAGGTGATACGTCCATGTAGTCCATTTTTTCT


 


TTAGCCATAACTGTGTTATTACCGCGGAAACGACAAACAACTTCGTCATC


 


TAAAAACTTACCATTTTCATCTAAGTTAGAGTTGGCTTGTGCTACCACAT


 


AGCTGTCCTCTTCATCAGCAGTTAGGTAATCAATTTGATCTGTAATTGAG


 


TTTGTTGCTAAATCTACTTTACGGTACGGCGTTTCGATAAAGCCAAATTC


 


ATTTACACGTGCATAACTTGATAGTGAGTTAATTAAACCGATGTTTGGTC


 


CCTCTGGTGTTTCGATAGGACACATACGGCCATAGTGAGAATAGTGTACG


 


TCACGTACTTCCATTTGAGCACGTTCACGTGTTAAACCACCAGGTCCTAA


 


TGCTGATAAACGACGTTTAT-3′






SEQ ID NO:39: Partial sequence of the rpoB gene in Staphylococcus caprae, measuring 556 base pairs:









(SEQ ID NO: 39)







5′TAACCCATTAGCTGAGTTGACTCATAAACGTCGTCTATCAGCATTAGG


 


ACCTGGTGGTTTAACGCGTGAACGTGCCCAAATGGAAGTGCGTGACGTTC


 


ACTATTCTCACTATGGCCGTATGTGTCCAATCGAAACACCTGAGGGACCA


 


AACATTGGTTTAATCAACTCATTATCAAGTTATGCACGAGTAAATGAATT


 


TGGTTTTATTGAAACACCTTATCGTAAAGTAGATTTAGATACGAATTCTA


 


TCACTGACCAAATTGATTACTTAACTGCTGATGAAGAAGATAGTTATGTT


 


GTTGCCCAAGCGAACTCTCGTTTAGACGAAAATGGTCGTTTCTTAGATGA


 


CGAAGTTGTTTGTCGTTTCCGTGGTAATAACACAGTTATGGCTAAAGAGA


 


AAATGGACTACATGGATGTATCTCCTAAACAAGTAGTATCTGCAGCGACA


 


GCTTGTATTCCATTCTTAGAAAATGATGACTCTAACCGTGCATTAATGGG


 


TGCGAACATGCAACGTCAAGCAGTACCATTGATGAATCCAGAAGCGCCAT


 


TTGTTGGT-3′






EXAMPLE 3
Blind Identification of a Collection of 20 Bacterial Strains Comprising 10 Strains of Bacteria Belonging to the Staphylococcus Genus
A collection of twenty strains belonging to the following bacterial species: Staphylococcus aureus (strain sensitive to rifampicin), Staphylococcus aureus (strain resistant to rifampicin), Staphylococcus epidermis, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus equorum, Staphylococcus schleiferi, Staphylococcus lugdunensis, Staphylococcus gallinarum, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Enterococcus faecalis, Streptococcus pyogenes, Corynebacterium amycolatum, Gemella morbilorum, Acinetobacter anitratus, Micrococcus luteus and Propionibacterium acnes was encoded so as to conduct blind molecular identification of the strains (the experimenter being unaware of strain identity) using the method described in this patent application. Extraction of the nucleic acids and amplification of the fragment of 751 base pairs of the rpoB gene were performed as described in example no 2 incorporating the primers SEQ ID NO:7 (as 5′ primer) and SEQ ID NO:10 (as 3′ primer) in a PCR amplification (FIG. 1). The sequencing of these 10 amplicons was conducted by incorporating within the sequencing reaction the primers SEQ ID NO:9 (5′ primer) and SEQ ID NO:10 (3′ primer) as described in example 2 and a comparison of the sequences obtained with the sequences of the data bank for sequences SEQ ID NOs:11 to 39 made it possible to identify the ten amplified strains as being: Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermis, 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 following the method that is the subject hereof and the identification established previously using standard phenotype methods. This result illustrates the specificity of the primer sets SEQ ID NO: 7/SEQ ID NO:10 and SEQ ID NO: 9/SEQ ID NO:10 used for this work and the fact that the level of sensitivity of Staphylococcus aureus strains to rifampicin does not interfere with the identification of these strains.
The other bacteria chosen because they are frequently isolated in human or animal clinical samples and also likely to contain bacteria of the Staphylococcus genus, were not amplified, thereby demonstrating the specificity of the primers used for the Staphylococcus genus under conditions of use to detect bacteria of the Staphylococcus genus using the method of the invention, relative to bacteria of another genus.
FIG. 1 shows the PCR amplification products obtained from fifteen encoded bacterial strains, comprising 10 strains belonging to the Staphylococcus genus (columns 2 to 5, 8, 9, 11 to 13 and 16) and 5 bacterial strains of bacterial geni other than Staphylococcus (columns 6, 7, 10, 14 and 15). Columns 1 and 17 represent the molecular weight labeler. Columns corresponding to negative amplification controls (sterile water) and to strains other than Staphylococcus are not shown. The amplification products are obtained after incorporating primers SEQ ID NO:7 and SEQ ID NO:10 according to the invention and are visualized by ethidium bromide staining after electrophoresis on agarose gel.

## Claims

1. A method of detecting a bacterium belonging to the Staphylococcus genus, the method comprising:
(a) contacting a sample containing a nucleic acid of a bacterium with amplification primers, the amplification primers comprising a 3′ primer and a 5′ primer;
(b) amplifying a nucleic acid present in the sample by an enzymatic polymerization reaction; and
(c) determining the presence of amplification product, wherein the presence of amplification product indicates the presence of a bacterium belonging to the Staphylococcus genus in the sample;
wherein:
the 3′ primer comprises at least one member selected from the group consisting of:
a primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:8;
a primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ primer comprises at least one member selected from the group consisting of:
a primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:9;
a primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount;

wherein the method indicates that a bacterium of the Staphylococcus genus is present in the sample if any species selected from the group consisting of 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 equorum, Staphylococcus epidermidis, Staphylococcus cohni, Staphylococcus chromogenes, Staphylococcus carnosus, Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus aureus subs. aureus, Staphylococcus aureus subs. anaerobius, Staphylococcus arlettae, and Staphylococcus caprae is present in the sample, with the exception that the primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:8 is not capable of detecting the presence of Staphylococcus schleiferi.

2. The method of claim 1, wherein:
the 3′ primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:8;
a primer comprising the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:9;
a primer comprising the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount.

3. The method of claim 1, wherein:
the 3′ primer comprises at least one primer comprising the sequence set forth in SEQ ID NO:10, where each n represents inosine; and
the 5′ primer comprises at least one primer comprising the sequence set forth in SEQ ID NO:7, where n represents inosine.

4. The method of claim 1, wherein:
the 3′ primer comprises at least one member selected from the group consisting of:
a primer consisting of the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ primer comprises at least one member selected from the group consisting of:
a primer consisting of the sequence set forth in SEQ ID NO:9;
a primer consisting of the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount.

5. The method of claim 4, wherein:
the 3′ primer comprises at least one primer consisting of the sequence set forth in SEQ ID NO:10, where each n represents inosine; and
the 5′ primer comprises at least one primer consisting of the sequence set forth in SEQ ID NO:7, where n represents inosine.

6. A method of detecting a species of bacterium belonging to the Staphylococcus genus, the species of the bacterium belonging to the group consisting of 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 equorum, Staphylococcus epidermidis, Staphylococcus cohni, Staphylococcus chromogenes, Staphylococcus carnosus, Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus aureus subs. aureus, Staphylococcus aureus subs. anaerobius, Staphylococcus arlettae, Staphylococcus caprae, the method comprising:
(a) performing a sequencing reaction on an amplified rpoB gene fragment of a bacterium using nucleotide sequencing primers, the nucleotide sequencing primers comprising a 3′ sequencing primer and a 5′ sequencing primer; and
(b) determining whether the bacterium belongs to a given species of the Staphylococcus genus by comparing the sequence of said fragment obtained with a sequence selected from the group consisting of SEQ ID NOs:11-39, and full-length complementary sequences thereof, wherein it is indicative that the bacterium belongs to the Staphylococcus species if the sequence obtained is identical to the sequence selected from the group consisting of SEQ ID NOs:11-39;
wherein:
the 3′ sequencing primer comprises at least one member selected from the group consisting of:
a primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ sequencing primer comprises at least one member selected from the group consisting of:
a primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:9;
a primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount.

7. The method of claim 6, wherein:
the 3′ sequencing primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ sequencing primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:9;
a primer comprising the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount.

8. The method of claim 6, wherein:
the 3′ sequencing primer is at least one primer consisting of the sequence set forth in SEQ ID NO:10, where each n represents inosine; and
the 5′ sequencing primer comprises at least one member selected from the group consisting of:
a primer consisting of the sequence set forth in SEQ ID NO:9; and
a primer consisting of the sequence set forth in SEQ ID NO:7, where n represents inosine.

9. The method of claim 6, wherein:
the amplified rpoB gene fragment is obtained by first amplification of a nucleic acid of said sample with a pair of 5′ and 3′ amplification primers, wherein:
the 5′ amplification primer comprises at least one member selected from the group consisting of:
a primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount, and

the 3′ amplification primer comprises at least one member selected from the group consisting of:
a primer comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each comprising at least 12 consecutive nucleotides of the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ sequencing primer comprises at least one primer comprising the sequence set forth in SEQ ID NO:9.

10. The method of claim 9, wherein:
the 3′ amplification primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ amplification primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount.

11. The method of claim 9, wherein:
the 3′ sequencing primer comprises at least one member selected from the group consisting of:
a primer consisting of the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ sequencing primer comprises at least one primer consisting of the sequence set forth in SEQ ID NO:9.

12. At least one isolated oligonucleotide or mixture of oligonucleotides selected from the group consisting of:
an oligonucleotide having no more than 35 nucleotides, the oligonucleotide comprising the sequence set forth in SEQ ID NO:8, or the full-length complementary sequence thereof;
an oligonucleotide having no more than 35 nucleotides, the oligonucleotide comprising the sequence set forth in SEQ ID NO:9, or of the full-length complementary sequence thereof;
an oligonucleotide having no more than 35 nucleotides, the oligonucleotide comprising the sequence set forth in SEQ ID NO:7, or the full-length complementary sequence thereof, where n represents inosine;
a mixture of oligonucleotides each having no more than 35 nucleotides and each comprising the sequence set forth in SEQ ID NO:7, or the full-length complementary sequence thereof, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount;
an oligonucleotide having no more than 35 nucleotides, the oligonucleotide comprising the sequence set forth in SEQ ID NO: 10, or the full-length complementary sequence thereof, where each n represents inosine;
a mixture of oligonucleotides each having no more than 35 nucleotides and each comprising the sequence set forth in SEQ ID NO: 10, or the full-length complementary sequence thereof, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount;
and a mixture of oligonucleotides each having no more than 35 nucleotides and each comprising the sequence set forth in SEQ ID NO: 10, or the full-length complementary sequence thereof, wherein:
n at nucleotide 3 of SEQ ID NO: 10 is inosine for each oligonucleotide of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO: 10 is inosine for each oligonucleotide of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO: 10 is inosine for each oligonucleotide of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture.

13. The at least one isolated oligonucleotide or mixture of oligonucleotides of claim 12, wherein said at least one oligonucleotide consists of a sequence selected from the group consisting of:
the sequence set forth in SEQ ID NO:7, or its full-length complementary sequence, where n is inosine;
the sequence set forth in SEQ ID NO:8, of its full-length complementary sequence thereof;
the sequence set forth in SEQ ID NO:9, of its full-length complementary sequence thereof; and
the sequence set forth in SEQ ID NO:10, or its full-length complementary sequence, where each n is inosine.

14. A method of detecting whether a sample contains any bacteria belonging to the Staphylococcus genus, the method comprising:
(a) contacting the sample with amplification primers, the amplification primers comprising a 3′ primer and a 5′ primer;
(b) amplifying a nucleic acid present in the sample by an enzymatic polymerization reaction; and
(c) determining the presence of amplification product, wherein the presence of amplification product indicates the presence of a bacterium belonging to the Staphylococcus genus in the sample;
wherein:
the 3′ primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:8;
a primer comprising the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:9;
a primer comprising the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount.

15. The method of claim 14, wherein:
the 3′ primer comprises at least one primer comprising the sequence set forth in SEQ ID NO:10, where each n represents inosine; and
the 5′ primer comprises at least one primer comprising the sequence set forth in SEQ ID NO:7, where n represents inosine.

16. The method of claim 14, wherein:
the 3′ primer comprises at least one member selected from the group consisting of:
a primer consisting of the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ primer comprises at least one member selected from the group consisting of:
a primer consisting of the sequence set forth in SEQ ID NO:9;
a primer consisting of the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount.

17. The method of claim 16, wherein:
the 3′ primer comprises at least one primer consisting of the sequence set forth in SEQ ID NO:10, where each n represents inosine; and
the 5′ primer comprises at least one primer consisting of the sequence set forth in SEQ ID NO:7, where n represents inosine.

18. A method of detecting a species of bacterium belonging to the Staphylococcus genus, the species of the bacterium belonging to the group consisting of 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 equorum, Staphylococcus epidermidis, Staphylococcus cohni, Staphylococcus chromogenes, Staphylococcus carnosus, Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus aureus subs. aureus, Staphylococcus aureus subs. anaerobius, Staphylococcus arlettae, Staphylococcus caprae, the method comprising:
(a) performing a sequencing reaction on an amplified rpoB gene fragment of a bacterium using nucleotide sequencing primers, the nucleotide sequencing primers comprising a 3′ sequencing primer and a 5′ sequencing primer; and
(b) determining whether the bacterium belongs to a given species of the Staphylococcus genus by comparing the sequence of said fragment obtained with a sequence selected from the group consisting of SEQ ID NOs:11-39, and full-length complementary sequences thereof, wherein it is indicative that the bacterium belongs to the Staphylococcus species if the sequence obtained is identical to the sequence selected from the group consisting of SEQ ID NOs:11-39;
wherein:
the 3′ sequencing primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ sequencing primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:9;
a primer comprising the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount.

19. The method of claim 18, wherein:
the amplified rpoB gene fragment is obtained by first amplification of a nucleic acid of said sample with a pair of 5′ and 3′ amplification primers, wherein:
the 3′ amplification primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ amplification primer comprises at least one member selected from the group consisting of:
a primer comprising the sequence set forth in SEQ ID NO:7, where n represents inosine; and
a mixture of primers each comprising the sequence set forth in SEQ ID NO:7, wherein n represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at that n in an equimolar amount; and

the 5′ sequencing primer comprises at least one primer comprising the sequence set forth in SEQ ID NO:9.

20. The method of claim 19, wherein:
the 3′ sequencing primer comprises at least one member selected from the group consisting of:
a primer consisting of the sequence set forth in SEQ ID NO:10, where each n represents inosine;
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:10, where each n independently represents A, T, C, or G and wherein, for the mixture, A, T, C, and G are represented at each n in an equimolar amount; and
a mixture of primers each consisting of the sequence set forth in SEQ ID NO:10, wherein:
n at nucleotide 3 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture;
n at nucleotide 6 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and
n at nucleotide 9 of SEQ ID NO:10 is inosine for each primer of the mixture or is represented by A, T, C, and G in equimolar amounts in the mixture; and


the 5′ sequencing primer comprises at least one primer consisting of the sequence set forth in SEQ ID NO:9.