DETECTION OF STAPHYLOCOCCUS AUREUS AND IDENTIFICATION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS

Abstract

Aspects of the present invention relate to methods and compositions for the detection and/or quantification of S. aureus from a sample, as well as methods and compositions useful for the detection and/or quantification of S. aureus and MRSA in a single assay. Embodiments include nucleic acids that hybridize to S. aureus-specific nuc sequences and MREJ sequences.

Description

RELATED APPLICATIONS

[0001] The present application is a continuation of an claims priority to U.S. patent application Ser. No. 11/959,337, filed Dec. 18, 2007, to Jean et al., entitled "DETECTION OF STAPHYLOCOCCUS AUREUS AND IDENTIFICATION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS," which claims priority to U.S. Provisional Application Ser. No. 60/870,823, filed on Dec. 19, 2006, to Jean et al. entitled "DETECTION OF STAPHYLOCOCCUS AUREUS AND IDENTIFICATION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS," the entire contents of which are hereby expressly incorporated by reference in their entireties.

SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled GENOM072C1.TXT, created Aug. 26, 2013, which is 129 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] Members of the genus Staphylococcus are major human pathogens, causing a wide variety of hospital and community acquired infections worldwide. The coagulase-positive species Staphylococcus aureus is well documented as a human opportunistic pathogen (Murray et al. Eds, 2003, Manual of Clinical Microbiology, 8th Ed., ASM Press, Washington, D.C.). Nosocomial infections caused by S. aureus are a major cause of morbidity and mortality. Some of the most common infections caused by S. aureus involve the skin, and they include furuncles or boils, cellulitis, impetigo, and postoperative wound infections at various sites. Some of the more serious infections produced by S. aureus are bacteremia, pneumonia, osteomyelitis, acute endocarditis, myocarditis, pericarditis, cerebritis, meningitis, scalded skin syndrome, and various abcesses. Food poisoning mediated by staphylococcal enterotoxins is another important syndrome associated with S. aureus. Toxic shock syndrome, a community-acquired disease, has also been attributed to infection or colonization with toxigenic S. aureus.

[0004] Coagulase-negative Staphylococci had been regarded as harmless skin commensals prior to the 1970s, however, they are now recognized as important causes of human infections (Kloos, et al. (2004) Clin. Microbiol. Rev. 7:117-140). In addition to being among the most frequently isolated bacteria in clinical microbiology laboratories, coagulase-negative Staphylococci serve as reservoirs of antimicrobial resistance determinants (Bastos, et al. (1999) Eur. J. Clin. Microbiol. Infect. Dis. 18:393-398). As such, it is important to characterize and distinguish S. aureus strains from other, coagulase-negative Staphylococci.

[0005] S. aureus strains produce an extracellular thermostable nuclease (thermostable TNase) with a frequency similar to that at which they produce coagulase. The sequence of the gene encoding TNase, nuc, was first disclosed in 1985 (Kovacevi et al. (1985), J. Bact. 162:521-528). TNase is a 17 kDa protein that degrades both RNA and DNA at temperatures up to 100° C. TNase activity is not specific for S. aureus, however, S. aureus species-specific sequences exist. See, e.g., Brackstad, et al. (1992), J. Clin. Microbiol. 30:1654-1660; Zhang, et al. (2004), J. Clin. Microbiol. 42:4947-4955; Chesneau, et al. (1993) Mol. Cell. Probes 7:301-310, Wilson, et al. (1991) Appl. Environ. Microbiol. 57:1793-1798; Poulsen et al., (2003) J. Antimicrob. Chemo. 51:419-421, Costa et al., (2005), Diag. Microbiol. and Infect. Dis, 51: 13-17, Shittu et al., (2006), Diagn Microbiol Infect Dis. 2006 Jul. 17. To date, none of the S. aureus-specific nuc sequences have been proven to be clinically useful by way of a large specificity study. Therefore, there exists a need for oligonucleotides that have been proven to be both highly specific and sensitive, which are useful in rapid detection and identification of S. aureus from clinical samples.

[0006] Both S. aureus and coagulase-negative Staphylococci have a remarkable ability to accumulate additional antibiotic resistant determinants, resulting in the formation of multidrug-resistant strains. This resistance limits therapeutic options for treatment and substantially increases patient morbidity and mortality. Methicillin-resistant S. aureus (MRSA) emerged in the 1980s as a major clinical and epidemiologic problem in hospitals (Oliveira et al., (2002) Lancet Infect Dis. 2:180-189). MRSA are resistant to all β-lactams including penicillins, cephalosporins, carbapenems, and monobactams, which are the most commonly used antibiotics to cure S. aureus infections. MRSA infections can only be treated with more toxic and more costly antibiotics, which are normally used as the last line of defense. Since MRSA can spread easily from patient to patient via personnel, hospitals over the world are confronted with the problem to control MRSA.

[0007] Methicillin resistance in S. aureus is unique in that it is due to acquisition of DNA from other coagulase-negative staphylococci (CNS), coding for a surnumerary β-lactam-resistant penicillin-binding protein (PBP), which takes over the biosynthetic functions of the normal PBPs when the cell is exposed to β-lactam antibiotics. S. aureus normally contains four PBPs, of which PBPs 1, 2 and 3 are essential. The low-affinity PBP in MRSA, termed PBP 2a (or PBP2'), is encoded by the choromosomal mecA gene and functions as a β-lactam-resistant transpeptidase. The mecA gene is absent from methicillin-sensitive S. aureus but is widely distributed among other species of staphylococci and is highly conserved (Ubukata et al., (1990) Antimicrob. Agents Chemother. 34:170-172).

[0008] Nucleotide sequence determination of the DNA region surrounding the mecA gene from S. aureus strain N315 (isolated in Japan in 1982), led to the discovery that the mecA gene is carried by a novel genetic element, designated staphylococcal cassette chromosome mec (SCCmec), which is inserted into the chromosome. SCCmec is a mobile genetic element characterized by the presence of terminal inverted and direct repeats, a set of site-specific recombinase genes (ccrA and ccrB), and the mecA gene complex (Ito et al., (1999) Antimicrob. Agents Chemother. 43:1449-1458; Katayama et al., (2000) Antimicrob. Agents Chemother. 44:1549-1555). SCCmec is precisely excised from the chromosome of S. aureus strain N315 and integrates into a specific S. aureus chromosomal site in the same orientation through recombinases encoded by the ccrA and ccrB genes. Cloning and sequence analysis of the DNA surrounding the mecA gene from MRSA strains NCTC 10442 (the first MRSA strain isolated in England in 1961) and 85/2082 (a strain from New Zealand isolated in 1985) led to the discovery of two novel genetic elements that shared similar structural features of SCCmec. The three SCCmec have been designated type I (NCTC 10442), type II (N315) and type III (85/2082) based on the year of isolation of the strains (Ito et al., (2001) Antimicrob. Agents Chemother. 45:1323-1336). Hiramatsu et al. have found that the SCCmec DNAs are integrated at a specific site in the chromosome of methicillin-sensitive S. aureus (MSSA). The nucleotide sequence of the regions surrounding the left and right boundaries of SCCmec DNA (i.e. attL and attR, respectively), as well as those of the regions around the SCCmec DNA integration site (i.e. attBscc which is the bacterial chromosome attachment site for SCCmec DNA), were analyzed. Sequence analysis of the attL, attR and attBscc sites revealed that attBscc is located at the 3' end of a novel open reading frame (ORF), orfX. orfX encodes a putative 159-amino acid polypeptide that exhibits sequence homology with some previously identified polypeptides of unknown function (Ito et al., (1999) Antimicrob. Agents Chemother. 43:1449-1458). Two new types of SCCmec, designated type IV and type V were recently described (Ma et al., (2002) Antimicrob. Agents Chemother. 46:1147-1152, Ito et al., (2004) Antimicrob Agents Chemother. 48:2637-2651, Oliveira et al., (2001) Microb. Drug Resist. 7:349-360). Oliveira et al. also recently reported the existence of SCCmec type VI. Oliveira et al., (2006), Antimicrob Agents Chemother. 50:3457-3459. The sequence of the right extremity of some Staphylococcus strains classified as SCCmec type IV has been determined. See, Ma et al., (2002) Antimicrob. Agents Chemother. 46:1147-1152; Ito et al., (2001) Antimicrob. Agents Chemother. 45:1323-1336; Oliveira et al., (2001) Microb. Drug Resist. 7:349-360. Sequences from S. aureus strains CA05 and 8/6-3P, classified as SCCmec type IV, were nearly identical over 2000 nucleotides to that of type II SCCmec of S. aureus strain N315 (Ma et al., (2002) Antimicrob. Agents Chemother. 46:1147-1152; Ito et al., (2001) Antimicrob. Agents Chemother. 45:1323-1336).

[0009] Methods to detect and identify MRSA based on the detection of the mecA gene and S. aureus-specific chromosomal sequences have been described. See, Schuenck et al., Res. Microbiol., (2006), in press, Shittu et al., (2006), Diagn Microbiol Infect Dis. July 17, Grisold et al., (2006), Methods Mol. Biol. 345: 79-89, Costa et al., (2005), Diag. Microbiol. and Infect. Dis, 51: 13-17, Mc Donald et al., (2005), J. Clin. Microbiol., 43: 6147-6149, Zhang et al., (2005), J. Clin. Microbiol. 43: 5026-5033, Hagen et al. (2005), Int J Med Microbiol. 295:77-86, Maes, et al. (2002) J. Clin. Microbiol. 40:1514-1517, Saito et al., (1995) J. Clin. Microbiol. 33:2498-2500; Ubukata et al., (1992) J. Clin. Microbiol. 30:1728-1733; Murakami et al., (1991) J. Clin. Microbiol. 29:2240-2244; Hiramatsu et al., (1992) Microbiol. Immunol. 36:445-453). Furthermore, Levi and Towner (2003), J. Clin. Microbiol., 41:3890-3892 and Poulsen et al. (2003), J Antimicrob Chemother., 51:419-421 describe detection of methicillin resistance in coagulase-negative Staphylococci and in S. aureus using the EVIGENE® MRSA Detection kit.

[0010] However, because the mecA gene is widely distributed in both S. aureus and coagulase-negative staphylococci, each of the methods described above are incapable of discriminating between samples containing both methicillin-sensitive S. aureus ("MSSA") and methicillin-resistant coagulase-negative staphylococci, and samples that contain only MRSA or that have both methicillin-sensitive S. aureus and MRSA.

[0011] To address this problem, Hiramatsu et al. developed a PCR-based assay specific for MRSA that utilizes primers that hybridize to the right extremities of DNA of SCCmec types I-III in combination with primers specific to the S. aureus chromosome, which corresponds to the nucleotide sequence on the right side of the SCCmec integration site. (U.S. Pat. No. 6,156,507, hereinafter the "'507 patent"). More recently, Zhang et al., (2005), J. Clin. Microbiol. 43: 5026-5033, described a multiplex assay for subtyping SCCmec types I to V MRSA. Nucleotide sequences surrounding the SCCmec integration site in other staphylococcal species (e.g., S. epidermidis and S. haemolyticus) are different from those found in S. aureus, therefore multiplex PCR assays that utilize oligonucleotides that hybridize to the right extremities of SCCmec and the S. aureus chromosome have the advantage of being specific for the detection of MRSA.

[0012] The PCR assay described in the '507 patent also led to the development of "MREP typing" (mec right extremity polymorphism) of SCCmec DNA (Ito et al., (2001) Antimicrob. Agents Chemother. 45:1323-1336; Hiramatsu et al., (1996) J. Infect. Chemother. 2:117-129). The MREP typing method takes advantage of the fact that the nucleotide sequences of the three MREP types differ at the right extremity of SCCmec DNAs adjacent to the integration site among the three types of SCCmec. Compared to type I, type III has a unique nucleotide sequence while type II has an insertion of 102 nucleotides to the right terminus of SCCmec. The MREP typing method described by Hiramatsu et al. uses the following nomenclature: SCCmec type I is MREP type i, SCCmec type II is MREP type ii, and SCCmec type III is MREP type iii. Hiramatsu later revised this nomenclature in view of the publication of the sequences of the genomes of strains N315 and Mu50, since the sequences revealed that SCCmec elements are located downstream of orfX. Consequently, MREP can now be referred to as MLEP (mec left extremity polymorphism) (Chongtrakool et al., (2006), Antimicrob. Agents Chemother. 50:1001-1012).

[0013] Recently, Chongtrakool et al. proposed replacing the SCCmec nomenclature with new nomenclature. Chongtrakool et al., (2006), Antimicrob. Agents Chemother. 50:1001-1012. Chongtrakool et al.'s proposed nomenclature is based on the structure of SCCmec elements and has three features. The first feature is a description of the SCC type and is defined by ccr type and mec class. The second feature is the description of the J regions (junkyard regions), which are part of the SCCmec element, located between and around the mec and ccr complexes. The third feature is the enumeration which allows the numbering of ccr type and J regions according to their time of identification.

[0014] As stated above, SCCmec types II and IV have the same nucleotide sequence to the right extremity. Consequently, the MREP (or MLEP according to recent revision) typing method described above cannot differentiate the SCCmec type IV described by Hiramatsu et al. (Ma et al., (2002) Antimicrob. Agents Chemother. 46:1147-1152) from SCCmec type II).

[0015] We recently described DNA sequences and regions in MRSA named MREJ. PCT Application No. PCT/CA02/00824. The phrase MREJ refers to the mec right extremity junction <<mec right extremity junction>>. MREJ's are approximately 1 kilobase (kb) in length and include sequences from the SCCmec right extremity as well as bacterial chromosomal DNA to the right of the SCCmec integration site. Importantly, MREJ sequences provide advantages over MREP/MLEP sequences in classifying MRSA in that MREJ/MLEJ sequences enable the differentiation between strains classified as SCCmec type II and SCCmec type IV. As discussed in PCT Application No. PCT/CA02/00824, the strains that Hiramatsu classified as MREP types i-iii fall under MREJ types i-iii according to the MREJ typing system. We recently identified novel MREJ types iv-xx, and developed nucleic acid assays with improved ubiquity capable of detection and identification of MRSA of MREJ types i-xx. (Huletsky et al., 2004, J. Clin. Microbiol. 42:1875-1884, International Patent Application PCT/CA02/00824, U.S. patent application Ser. No. 11/248,438). Based on the revision of MREP to MLEP, one can understand that previously called MREJ types could now be reclassified as MLEJ (mec left extremity junction). The skilled artisan will appreciate that any S. aureus and MRSA classification system is contemplated in the methods disclosed herein, as sequences can specifically detect S. aureus and identify those which are resistant to methicillin.

[0016] Maes et al. describe a PCR assay to discriminate S. aureus from coagulase negative Staphylococci and to determine methicillin resistance in blood cultures (Maes, et al. (2002) J. Clin. Microbiol. 40:1514-1517). The assay described in Maes et al. cannot distinguish MRSA from methicillin-resistant coagulase-negative Staphylococci.

[0017] Poulsen et al. describe detection of methicillin resistance in coagulase-negative Staphylococci and in S. aureus using the EVIGENE® MRSA Detection kit. The assay described in Poulsen et al. cannot discriminate between a sample that has both methicillin-sensitive S. aureus and methicillin-resistant coagulase-negative staphylococci, and a sample that contains only MRSA or that has both methicillin-sensitive S. aureus and MRSA.

[0018] Accordingly, there remains a need for a rapid assay to detect and identify both MRSA and methicillin-sensitive S. aureus in the same reaction and to be able to distinguish S. aureus from coagulase-negative Staphylococci in the same reaction.

SUMMARY OF THE INVENTION

[0019] Disclosed herein are methods and compositions for specifically detecting the presence of a Staphylococcus aureus (S. aureus) strain and detecting the presence of a methicillin-resistant S. aureus (MRSA) strain from a clinical sample in a single assay. Also provided herein are methods and compositions for the specific detection of S. aureus from a sample.

[0020] Some embodiments relate to methods of detecting S. aureus and identifying the presence of MRSA from a sample that includes nucleic acids. In some embodiments, the sample can be contacted with at least one primer and or probe of at least 10 nucleotides that anneals under stringent conditions a S. aureus-specific sequence of the nuc gene, and at least one primer and/or probe specific for a MRSA strain. S. aureus strains are rendered methicillin-resistant due to the presence of an SCCmec cassette containing a mecA gene that is inserted in bacterial nucleic acids. The insertion of the SCCmec cassette can generate a polymorphic right extremity junction (MREJ). The MRSA-specific primer(s) and/or probe(s) can anneal under stringent conditions to polymorphic MREJ nucleic acids, including, for example, MREJ types i to xx. S. aureus-specific and MRSA-specific primers anneal under conditions of, for example, 4 mM MgCl₂, 100 mM Tris (pH 8.3), 10 mM KCl, and 5 mM (NH₄)₂SO₄ at 59° C. The presence and/or amount of annealed probe(s), or amplification products produced through annealing of the primers to the nucleic acids, can be used as an indication of the presence and/or amount of S. aureus (MSSA and MRSA) and MRSA in the sample.

[0021] The at least one primer specific for a S. aureus strain can anneal under stringent conditions to the SEQ ID NO: 200, the complement thereof or any sequence which differs from SEQ ID NO: 200 by 1 to 20 nucleotides.

[0022] In some embodiments, the at least one primer and/or probe that anneals under stringent conditions to the S. aureus specific nuc sequence hybridizes under stringent conditions to one of the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or the complement thereof. Preferably, the at least one primer and/or probe that anneals under stringent conditions to the S. aureus specific nuc sequence comprises, consists essentially of, or consists of one of the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0023] In preferred embodiments, the S. aureus-specific primer(s) and/or probe(s) are at least 10 nucleotides in length, and anneal under stringent conditions to the nucleic acid of any one of SEQ ID NOs: 1 to 12 or the complement thereof.

[0024] In still more preferred embodiments, the sample is also contacted with a probe that anneals under stringent conditions to the nucleic acid of any one of SEQ ID NOs: 9, 10, 11, or 12, or the complement thereof. In some embodiments, the probe is a molecular beacon probe. Preferably, the probe comprises, consists essentially of, or consists of the sequence of SEQ ID NOs: 9, 10, 11, or 12.

[0025] In some embodiments, the method also includes adding internal control DNA to the sample, and at least one primer and/or probe that anneals under stringent conditions to the internal control DNA. For example, in some embodiments, the Internal Control can be a linearized 4.23 kb plasmid purified from E. coli. The internal control can be used to monitor the presence of inhibitory substances coming from a specimen.

[0026] The at least one primer specific for an MRSA strain can anneal under stringent conditions to the MREJ sequences of types i to xx, as defined in any one of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88, the complement thereof or any sequence which differs from SEQ ID NOs 14 to 88 by 1 to 20 nucleotides.

[0027] In preferred embodiments, S. aureus-specific and MRSA-specific primers and/or probes are chosen to anneal to the sample nucleic acids under the same annealing conditions. In more preferred embodiments, the primer(s) and/or probe(s) are placed altogether in the same physical enclosure.

[0028] In preferred embodiments, the MRSA-specific primer(s) and/or probe(s) are at least 10 nucleotides in length, and anneal under stringent conditions to the nucleic acid of any one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201 (types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, 198 (types x-xx) or 199 or the complement thereof. Preferably, the MRSA-specific primer(s) and/or probe(s) comprise, consist essentially of, or consist of the nucleic acid of any one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201 (types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, (types xi-xx) or 199. In some embodiments, MRSA-specific primers also include an oligonucleotide that hybridizes under stringent condition to orf22 of the S. aureus chromosome, wherein the primer can be used in an amplification reaction with SEQ ID NO: 197 to detect MREJ type x. In more preferred embodiments, the MRSA-specific primer(s) and/or probe(s) anneal under stringent conditions to the nucleic acid of any one of SEQ ID NOs: 99, 199, 144, 150, 155, and 163 or the complement thereof, such as a primer and/or probe that comprises, consists essentially of, or consists of the nucleic acid of any one of SEQ ID NOs: 99, 199, 144, 150, 155, and 163. In still more preferred embodiments, the sample is also contacted with a probe that anneals under stringent conditions to the nucleic acid of any one of SEQ ID NOs: 126, 128, 130 and 131, or the complement thereof. In some embodiments, the probe is a molecular beacon probe. Preferably, the probe comprises, consists essentially of, or consists of the nucleic acid of any one of SEQ ID NOs: 126, 128, 130 and 131.

[0029] In some embodiments, the sample and primer(s) and/or probe(s) described above are used in an amplification reaction, such as a PCR, LCR, NABSA, 3SR, SDA, bDNA, TMA, CPT, SPA, NDSA, rolling circle amplification, anchored-strand displacement amplification, solid-phase (immobilized) rolling circle amplification, or Q beta replicase amplification reaction.

[0030] Other aspects relate to the specific detection of an S. aureus strain in a sample that includes nucleic acids. At least one primer and/or probe that is specific for the nuc gene of S. aureus is provided. The primers and/or probe(s) include a nucleic acid that can anneal to at least 11 consecutive nucleotides of any one of the nucleic acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or the complement thereof, under stringent conditions, such as 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂ at 59° C.; or 4 mM MgCl₂, 100 mM Tris (pH 8.3), 10 mM KCl, and 5 mM (NH₄)₂SO₄ at 59° C. The primer(s) and/or probe(s) are allowed to anneal to the nucleic acids of the sample. Annealed primer(s) and/or probe(s) indicate the presence of an S. aureus strain in the sample. The annealed primer(s) and/or probe(s) can be detected, and the presence and/or amount of annealed probe(s), the amount of an amplification product produced through annealing of the primers to the nucleic acids, indicates the presence and/or amount of S. aureus present in the sample.

[0031] In some embodiments, the sample and primer(s) and/or probe(s) described above are used in an amplification reaction, such as a PCR, LCR, NABSA, 3SR, SDA, bDNA, TMA, CPT, SPA, NDSA, rolling circle amplification, anchored-strand displacement amplification, solid-phase (immobilized) rolling circle amplification, or Q beta replicase amplification reaction.

[0032] In preferred embodiments, a primer pair including a first primer that anneals under stringent conditions to SEQ ID NO:1 or the complement thereof (such as a primer that comprises, consists essentially of, or consists of SEQ ID NO: 1), and a second primer that anneals under stringent conditions to SEQ ID NO: 6 (such as a primer that comprises, consists essentially of, or consists of SEQ ID NO: 6), or the complement thereof, is allowed to anneal to the nucleic acids of the sample. In more preferred embodiments, a probe that anneals under stringent conditions to SEQ ID NO: 9 or 10 (such as a probe that comprises, consists essentially of, or consists of SEQ ID NO: 9 or 10), or the complement thereof, is also provided.

[0033] In other preferred embodiments, a primer pair including a first primer that anneals under stringent conditions to SEQ ID NO: 3 (such as a primer that comprises, consists essentially of, or consists of SEQ ID NO: 3) or the complement thereof, and a second primer that anneals under stringent conditions to SEQ ID NO: 8 (such as a primer that comprises, consists essentially of, or consists of SEQ ID NO: 8) or the complement thereof, is allowed to anneal to the nucleic acids of the sample. In more preferred embodiments, a probe that anneals under stringent conditions to SEQ ID NO: 11 or 12, (such as a primer that comprises, consists essentially of, or consists of SEQ ID NO: 11 or 12) or the complement thereof, is also provided.

[0034] In still other preferred embodiments, the sample is contacted with at least one primer pair, that includes a first primer and a second primer that anneal under stringent conditions to the nucleic acid sequence of at least one of the following pairs:

[0035] SEQ ID NOs: 1 and 5;

[0036] SEQ ID NOs: 1 and 6;

[0037] SEQ ID NOs: 2 and 5;

[0038] SEQ ID NOs: 2 and 6;

[0039] SEQ ID NOs: 3 and 7

[0040] SEQ ID NOs: 3 and 8;

[0041] SEQ ID NOs: 4 and 7; and

[0042] SEQ ID NOs: 4 and 8, or the complements thereof.

[0043] For example, in preferred embodiments, the sample is contacted with at least one primer pair that includes a first primer and a second primer that comprise, consist essentially of, or consist of the nucleic acid sequence of at least one of the following pairs:

[0044] SEQ ID NOs: 1 and 5;

[0045] SEQ ID NOs: 1 and 6;

[0046] SEQ ID NOs: 2 and 5;

[0047] SEQ ID NOs: 2 and 6;

[0048] SEQ ID NOs: 3 and 7

[0049] SEQ ID NOs: 3 and 8;

[0050] SEQ ID NOs: 4 and 7; and

[0051] SEQ ID NOs: 4 and 8.

[0052] In preferred embodiments, the sample is also contacted with at least one primer pair including a first primer and a second primer that anneal under stringent conditions to the nucleic acid sequence of at least one of the following pairs:

[0053] SEQ ID NOs: 99 and 199;

[0054] SEQ ID NOs: 99 and 144;

[0055] SEQ ID NOs: 99 and 150;

[0056] SEQ ID NOs: 99 and 155; and

[0057] SEQ ID NOs: 99 and 163, or the complement thereof.

[0058] For example, in some embodiments, the sample is contacted with at least one primer pair including a first primer and a second primer that comprise, consist essentially of, or consist of at least one of the following pairs:

[0059] SEQ ID NOs: 99 and 199;

[0060] SEQ ID NOs: 99 and 144;

[0061] SEQ ID NOs: 99 and 150;

[0062] SEQ ID NOs: 99 and 155; and

[0063] SEQ ID NOs: 99 and 163.

[0064] In preferred embodiments, the sample is contacted with a plurality of primer pairs, wherein the primers anneal under stringent conditions to the nucleic acid sequences of

[0065] SEQ ID NOs: 1 and 6

[0066] SEQ ID NOs: 99 and 199;

[0067] SEQ ID NOs: 99 and 144;

[0068] SEQ ID NOs: 99 and 150;

[0069] SEQ ID NOs: 99 and 155; and

[0070] SEQ ID NOs: 99 and 163, or the complements thereof, such as primer pairs that comprise, consist essentially of, or consist of the nucleic acid sequences of:

[0071] SEQ ID NOs: 1 and 6

[0072] SEQ ID NOs: 99 and 199;

[0073] SEQ ID NOs: 99 and 144;

[0074] SEQ ID NOs: 99 and 150;

[0075] SEQ ID NOs: 99 and 155; and

[0076] SEQ ID NOs: 99 and 163.

[0077] In other preferred embodiments, the sample is contacted with a plurality of primer pairs, wherein the primers anneal under stringent conditions to the nucleic acid sequences of

[0078] SEQ ID NOs: 3 and 8

[0079] SEQ ID NOs: 99 and 199;

[0080] SEQ ID NOs: 99 and 144;

[0081] SEQ ID NOs: 99 and 150;

[0082] SEQ ID NOs: 99 and 155; and

[0083] SEQ ID NOs: 99 and 163, or the complements thereof, such as primer pairs that comprise, consist essentially of, or consist of the nucleic acid sequences of:

[0084] SEQ ID NOs: 3 and 8

[0085] SEQ ID NOs: 99 and 199;

[0086] SEQ ID NOs: 99 and 144;

[0087] SEQ ID NOs: 99 and 150;

[0088] SEQ ID NOs: 99 and 155; and

[0089] SEQ ID NOs: 99 and 163.

[0090] Preferably, the sample is also contacted with at least one probe that anneals under stringent conditions to the nucleic acid sequence of any one of SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 and 131 or the complement thereof, such as a probe that comprises, consists essentially of, or consists of the nucleic acid sequence of any one of SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 and 131.

[0091] Other aspects relate to oligonucleotides useful for the specific detection of S. aureus. Some embodiments provide oligonucleotides which anneal under stringent conditions with at least 11 consecutive nucleotides of the nucleic acid sequence of one of the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, such as nucleic acids that comprise, consist essentially of, or consist of one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0092] Yet other aspects relate to kits for detecting the presence of an S. aureus strain in a sample that includes nucleic acids. The kit can include at least one oligonucleotide that anneals under stringent conditions to one of the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, or the complement thereof. For example, the kit can include at least one oligonucleotide that comprises, consists essentially of, or consists of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In preferred embodiments, the kit also includes at least one probe, wherein the probe can anneal to the nucleic acid sequence of SEQ ID NO: 9, 10, 11 or 12, or the complement thereof, under stringent conditions. In preferred embodiments, the probe can comprise, consist essentially of, or consist of SEQ ID NO: 9, 10, 11 or 12.

[0093] In preferred embodiments, the kit also includes at least one primer specific for an MRSA strain. S. aureus strains are rendered methicillin-resistant due to the presence of an SCCmec insert containing a mecA gene that is inserted in bacterial nucleic acids. The insertion of the SCCmec insert can generate a polymorphic right extremity junction (MREJ). The MRSA-specific primer(s) and/or probe(s) can anneal under stringent conditions to polymorphic MREJ nucleic acids, including, for example, MREJ types i to xx.

[0094] In preferred embodiments, the kit includes at least one MRSA-specific oligonucleotide that anneals under stringent conditions to one of the following SEQ ID NOs: SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88, or the complement thereof. For example, in some embodiments, a kit can contain at least one MRSA-specific oligonucleotide that is at least 10 nucleotides in length, and anneals under stringent conditions to the nucleic acid sequence of any one of the following SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201 (types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, (types xi-xx) or 199 or the complement thereof. In some embodiments, the MRSA-specific oligonucleotides can also include an oligonucleotide that hybridizes under stringent conditions to orf22 of the S. aureus chromosome, wherein the oligonucleotide can be used in an amplification reaction with SEQ ID NO: 197 to detect MREJ type x. Preferably, the MRSA-specific oligonucleotide can comprise, consist essentially of, or consist of any one of the following SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201 (types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, (types xi-xx) or 199.

[0095] In some embodiments, the kit contains a plurality of oligonucleotides that anneal under stringent conditions to SEQ ID NOs: 1, 6, 99, 144, 150, 155, and 163. For example, in preferred embodiments, the kit can contain a plurality of oligonucleotides that comprise, consist essentially of, or consist of SEQ ID NOs: 1, 6, 99, 144, 150, 155, and 163. In some embodiments, the kit contains a plurality of oligonucleotides that anneal under stringent conditions to SEQ ID NOs: 3, 8, 99, 144, 150, 155, and 163, such as a plurality of oligonucleotides that comprise, consist essentially of, or consist of SEQ ID NOs: 3, 8, 99, 144, 150, 155, and 163. Preferably, the kit also includes at least one probe that anneals under stringent conditions to the following SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 or 131, such as at least one probe that comprises, consists essentially of, or consists of SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 or 131.

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] FIGS. 1A and 1B show photographs of agarose gels showing the products of PCR amplification reactions. The number of copies and source of template DNA are indicated (15 cp=15 copies; 185 cp=185 copies) (S. aureus=MSSA strain ATCC 25923; MRSA=MRSA strain ATCC 43300). Arrows indicate the PCR product sizes and primer dimers.

[0097] FIGS. 2A and 2B show a graphical depiction of PCR amplification curves measured from reactions containing molecular beacon probes. Reactions contained 0, 2.5, 5, 10, 15, or 20 copies of MSSA (FIG. 2A) or MRSA (FIG. 2B) template DNA, as well as 3000 copies of internal control DNA. Molecular beacon probes were added to each reaction and the fluorescence of the reactions was measured. FAM labeled probes hybridize to MRSA-specific sequences, TET-labeled probes hybridize to internal control DNA sequences, and Texas-Red-labeled probes hybridize to S. aureus-specific nuc sequences.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] Methods and compositions disclosed herein relate to detection and/or quantification of S. aureus in a sample, and also relate to detection and/or quantification of a Staphylococcus aureus (S. aureus) strain and identification a methicillin-resistant S. aureus strain from a sample in a single assay. The embodiments disclosed herein are useful for detection and/or quantification of S. aureus and MRSA from any type of sample, such as any clinical sample, any environmental sample, any microbial culture, any microbial colony, any tissue, and any cell line.

[0099] Staphylococci are Gram-positive cocci. S. aureus can be distinguished from other clinically relevant species of Staphylococcus by a positive result on the basis of their ability to clot blood plasma (the coagulase reaction) and their ability to form clumps in the presence of fibrinogen. S. aureus, as some other staphylococci has the ability to produce a thermostable nuclease (TNase), Becker et al., (2005), Diagn Microbiol Infect Dis., 51:237-244, Brakstad et al, (1995), APMIS, 103:219-224, Chesneau, et al. (1993) Mol. Cell. Probes 7:301-310. Nevertheless, some nucleotide sequences in the gene encoding the nuclease are specific of S. aureus strains (Costa et al., (2005), Diag. Microbiol. and Infect. Dis, 51: 13-17, Mc Donald et al., (2005), J. Clin. Microbiol., 43: 6147-6149, Zhang, et al. (2004), J. Clin. Microbiol. 42:4947-4955; Maes, et al. (2002) J. Clin. Microbiol. 40:1514-1517).

Methods of Detecting S. aureus or S. aureus and MRSA

[0100] Some embodiments relate to methods of specifically detecting S. aureus in a sample. Disclosed herein are novel primers and/or probes (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) that anneal to S. aureus-specific sequences of the nuc gene, exemplified by SEQ ID NO: 200, which are useful to distinguish S. aureus from other Staphylococci, as well as other TNase-producing species of bacteria. In some embodiments, at least one primer and/or probe that anneals under stringent conditions to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or the complement thereof is provided. For example, in some embodiments, the at least one primer and/or probe can comprise, consist essentially of, or consist of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. The at least one primer is allowed to anneal to the nucleic acids of the sample, e.g., under standard PCR conditions or stringent conditions. The presence and/or amount of annealed probe(s) and/or the amount of an amplification product produced through annealing of the primers to the nucleic acids, is detected, thereby indicating the presence and/or amount of S. aureus present in the sample.

[0101] The term "consisting essentially of," when used in reference to nucleic acid can refer to the specified nucleic acid sequences, and can include any additional nucleotide that does not materially affect the basic and novel characteristics of the specified sequence. The term "consisting essentially of" also can refer to variants that are substantially similar to, and differ from a reference sequence in an inconsequential way as judged by examination of the sequence. For example, nucleic acid sequences encoding the same amino acid sequence are substantially similar despite differences in degenerate positions or modest differences in length or composition of any non-coding regions.

Primers and/or Probes and Nucleotides

[0102] As used herein, the terms "primer" and "probe" are not limited to oligonucleotides or nucleic acids, but rather encompass molecules that are analogs of nucleotides, as well as nucleotides. Nucleotides and polynucleotides, as used herein shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as NEUGENE® polymers), and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.

[0103] The terms nucleotide and polynucleotide include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3'→P5' phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA. The terms also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.

[0104] It will be appreciated that, as used herein, the terms "nucleoside" and "nucleotide" will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides will also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with a halogen, an aliphatic group, or are functionalized as ethers, amines, or the like. Other modifications to nucleotides or polynucleotides involve rearranging, appending, substituting for, or otherwise altering functional groups on the purine or pyrimidine base which form hydrogen bonds to a respective complementary pyrimidine or purine. The resultant modified nucleotide or polynucleotide may form a base pair with other such modified nucleotidic units but not with A, T, C, G or U. For example, guanosine (2-amino-6-oxy-9-beta.-D-ribofuranosyl-purine) may be modified to form isoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Such modification results in a nucleoside base which will no longer effectively form a standard base pair with cytosine. However, modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine) results in a modified nucleotide which will not effectively base pair with guanosine but will form a base pair with isoguanosine. Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine may be prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and references cited therein; 2'-deoxy-5-methyl-isocytidine may be prepared by the method of Tor et al. (1993) J. Am. Chem. Soc. 115:4461-4467 and references cited therein; and isoguanine nucleotides may be prepared using the method described by Switzer et al. (1993), supra, and Mantsch et al. (1993) Biochem. 14:5593-5601, or by the method described U.S. Pat. No. 5,780,610 to Collins et al. The non-natural base pairs referred to as κ and π., may be synthesized by the method described in Piccirilli et al. (1990) Nature 343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement (1-methylpyrazolo[4,3]-pyrimidine-5,7-(4H,6H)-dione. Other such modified nucleotidic units which form unique base pairs have been described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra, or will be apparent to those of ordinary skill in the art.

[0105] Primers and/or probes are preferably between 10 and 45 nucleotides in length. For example, the primers and or probes can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more nucleotides in length. Primers and/or probes can be provided in any suitable form, included bound to a solid support, liquid, and lyophilized, for example.

Annealing and Specific Binding

[0106] Binding or annealing of the primers and/or probes to target nucleic acid sequences is accomplished through hybridization. It will be appreciated by one skilled in the art that specific hybridization is achieved by selecting sequences which are at least substantially complementary to the target or reference nucleic acid sequence. This includes base-pairing of the oligonucleotide target nucleic acid sequence over the entire length of the oligonucleotide sequence. Such sequences can be referred to as "fully complementary" with respect to each other. Where an oligonucleotide is referred to as "substantially complementary" with respect to a nucleic acid sequence herein, the two sequences can be fully complementary, or they may form mismatches upon hybridization, but retain the ability to hybridize under stringent conditions or standard PCR conditions as discussed below.

[0107] A positive correlation exists between probe length and both the efficiency and accuracy with which a probe will anneal to a target sequence. In particular, longer sequences have a higher melting temperature (T_m) than do shorter ones, and are less likely to be repeated within a given target sequence, thereby minimizing promiscuous hybridization.

[0108] As used herein, "T_m" and "melting temperature" are interchangeable terms which refer to the temperature at which 50% of a population of double-stranded polynucleotide molecules becomes dissociated into single strands. Formulae for calculating the T_m of polynucleotides are well known in the art. For example, the T_m may be calculated by the following equation: T_m=69.3+0.41 x.(G+C) %-6-50/L, wherein L is the length of the probe in nucleotides. The T_m of a hybrid polynucleotide may also be estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used for calculating T_m for PCR primers: [(number of A+T)×2° C.+(number of G+C)×4° C.]. See, e.g., C. R. Newton et al. PCR, 2nd Ed., Springer-Verlag (New York: 1997), p. 24. Other more sophisticated computations exist in the art, which take structural as well as sequence characteristics into account for the calculation of T_m. A calculated T_m is merely an estimate; the optimum temperature is commonly determined empirically.

[0109] Primer or probe sequences with a high G+C content or that comprise palindromic sequences tend to self-hybridize, as do their intended target sites, since unimolecular, rather than bimolecular, hybridization kinetics are generally favored in solution. However, it is also important to design a probe that contains sufficient numbers of G:C nucleotide pairings since each G:C pair is bound by three hydrogen bonds, rather than the two that are found when A and T (or A and U) bases pair to bind the target sequence, and therefore forms a tighter, stronger bond. Preferred G+C content is about 50%.

[0110] Hybridization temperature varies inversely with probe annealing efficiency, as does the concentration of organic solvents, e.g., formamide, which might be included in a hybridization mixture, while increases in salt concentration facilitate binding. Under stringent annealing conditions, longer hybridization probes, or synthesis primers, hybridize more efficiently than do shorter ones, which are sufficient under more permissive conditions. Preferably, stringent hybridization is performed in a suitable buffer under conditions that allow the reference or target nucleic acid sequence to hybridize to the probes. Stringent hybridization conditions can vary (for example from salt concentrations of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM) and hybridization temperatures can range (for example, from as low as 0° C. to greater than 22° C., greater than about 30° C. and (most often) in excess of about 37° C. depending upon the lengths and/or the nucleic acid composition of the probes. Stringent hybridization temperatures for PCR range from 40 and 75° C., preferably between 45 and 70° C., depending on lengths and compositions of primers. Longer fragments may require higher hybridization temperatures for specific hybridization. As several factors affect the stringency of hybridization, the combination of parameters is more important than the absolute measure of a single factor. Accordingly, by way of example, the term "stringent hybridization conditions" may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). For example, the term "stringent conditions" encompasses standard PCR conditions, as described below.

[0111] For a review of PCR technology, including standard PCR conditions, applied to clinical microbiology, see DNA Methods in Clinical Microbiology, Singleton P., published by Dordrecht; Boston: Kluwer Academic, (2000) Molecular Cloning to Genetic Engineering White, B. A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997) and "PCR Methods and Applications", from 1991 to 1995 (Cold Spring Harbor Laboratory Press). Non-limiting examples of "PCR conditions" include the conditions disclosed in the references cited herein, and also in the examples below, such as, for example, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, with an annealing temperature of 72° C.; or 4 mM MgCl₂, 100 mM Tris, pH 8.3, 10 mM KCl, 5 mM (NH₄)₂SO₄, 0.15 mg BSA, 4% Trehalose, with an annealing temperature of 59° C., or 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, with an annealing temperature of 55° C.

[0112] As used herein, when used to describe primers and/or probes, the terms "specific" or "species-specific" refer to primers and/or probes which hybridize or anneal under stringent conditions and/or standard PCR conditions to nucleic acids of a specified species or type (e.g. S. aureus or MRSA), and which do not substantially anneal or hybridize under the same conditions to unrelated nucleic acids, such as nucleic acids other than the specified species or MREJ type.

[0113] In a preferred embodiment, the probes or primers described herein hybridize under stringent conditions to target sequences (e.g., S. aureus specific nuc sequences or MREJ sequences). In other preferred embodiments, the primers or probes described herein exhibit 100% complementarity over at least 10 to 45 nucleotides in length. For example, the primers and or probes exhibit complementarity over at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more nucleotides to the target sequence. In some embodiments, the primers or probes exhibit 100% complementarity to the target sequence over 10 to 45 consecutive nucleotides in all but at least 1 position (e.g., the primer and/or probe contains a mismatch), 2 positions, 3 positions, 4 positions, 5 positions, 6 positions, 7 positions or more.

[0114] Probes or primers that include sequences that can hybridize as described herein and that also include a portion that does not hybridize to the target sequence (e.g., a tag or a marker), are also contemplated. For example, in some embodiments, the primer and/or probe can contain a detectable moiety, such as a fluorescent moiety, or any other detectable marker, such as those described below. In some embodiments, the primer and/or probe may contain nucleic acid or other molecular components that facilitate subsequent manipulations, such as polymerization reactions, or enzymatic reactions such as digestion with restriction endonucleases, and the like, or that couple the primer and/or probe to a solid support.

Amplification and Detection

[0115] In the methods described herein, detection of annealed primers and/or probes can be direct or indirect. For example, probes can be annealed to the sample being tested, and detected directly. On the other hand, primers can be annealed to the sample being tested, followed by an amplification step. The amplified products can be detected directly, or through detection of probes that anneal to the amplification products.

[0116] In preferred embodiments, an amplification and/or detection step follows the annealing step. In other preferred embodiments, detection occurs during the annealing step. Any type of nucleic acid amplification technology can be used in the methods described herein. Non-limiting examples of amplification reactions that can be used in the methods described herein include but are not restricted to: polymerase chain reaction (PCR) (See, PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y. (Innis)), ligase chain reaction (LCR) (See, Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR) (See, Guatelli (1990) Proc. Natl. Acad. Sci. USA, 87:1874), strand displacement amplification (SDA), branched DNA signal amplification bDNA, transcription-mediated amplification (TMA) (See, Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173), cycling probe technology (CPT), nested PCR, multiplex PCR, solid phase amplification (SPA), nuclease dependent signal amplification (NDSA), rolling circle amplification technology (RCA), Anchored strand displacement amplification, solid-phase (immobilized) rolling circle amplification, Q Beta replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario). These and other techniques are also described in Berger (1987) Methods Enzymol. 152:307-316; Sambrook, Ausubel, Mullis (1987) U.S. Pat. Nos. 4,683,195 and 4,683,202; Amheim (1990) C&EN 36-47; Lomell J. Clin. Chem., 35:1826 (1989); Van Brunt, Biotechnology, 8:291-294 (1990); Wu (1989) Gene 4:560; Sooknanan (1995) Biotechnology 13:563-564.

[0117] In preferred embodiments, PCR is used to amplify nucleic acids in the sample. During DNA amplification by PCR, two oligonucleotide primers binding respectively to each strand of the heat-denatured target DNA from the microbial genome, are used to amplify exponentially in vitro the target DNA. Successive thermal cycles allow denaturation of the DNA, annealing of the primers and synthesis of new targets at each cycle. (Persing et al, (1993), Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).

[0118] The skilled artisan will appreciate that standard amplification protocols may be modified to improve nucleic acid amplification efficiency, including modifications to the reaction mixture. (Ralser et al., (2006), Biochem Biophys Res Commun., 347:747-51, Kang et al., (2005), J Biochem Biophys Methods. (2005), 64:147-51, Chakrabarti and Schutt, (2002), Biotechniques, 32:866-874; Al-Soud and Radstrom, (2000), J. Clin. Microbiol., 38:4463-4470; Al-Soud and Radstrom, 1998, Appl. Environ. Microbiol., 64:3748-3753; Wilson, 1997, Appl. Environ. Microbiol., 63:3741-3751). Such modifications of the amplification reaction mixture include but are not limited to the use of various polymerases or the addition of nucleic acid amplification facilitators such as betaine, BSA, sulfoxides, protein gp32, detergents, cations, and tetramethylamonium chloride.

[0119] Detection of amplified nucleic acids may include any real-time or post-amplification technologies known to those skilled in the art. Classically, the detection of PCR amplification products is performed by standard ethidium bromide-stained agarose gel electrophoresis, however, the skilled artisan will readily appreciate that other methods for the detection of specific amplification products, which may be faster and more practical for routine diagnosis, may be used, such as those described in co-pending patent application WO01/23604 A2. Amplicon detection may also be performed by solid support or liquid hybridization using species-specific internal DNA probes hybridizing to an amplification product. Such probes may be generated from any sequence from the MREJ or nuc nucleic acid sequences provided herein, and designed to specifically hybridize to DNA amplification products produced utilizing the methods disclosed herein. Alternatively, amplicons can be characterized by sequencing. See, e.g., co-pending patent application WO01/23604 A2 for examples of detection and sequencing methods.

[0120] Other non-limiting examples of nucleic acid detection technologies that can be used in the embodiments disclosed herein include, but are not limited to the use of fluorescence resonance energy transfer (FRET)-based methods such as adjacent hybridization of probes (including probe-probe and probe-primer methods) (See, J. R. Lakowicz, "Principles of Fluorescence Spectroscopy," Kluwer Academic/Plenum Publishers, New York, 1999)., TaqMan probe technology (See, European Patent EP 0 543 942), molecular beacon probe technology (See, Tyagi et al., (1996) Nat. Biotech. 14:303-308.), Scorpion probe technology (See, Thewell (2000), Nucl. Acids Res. 28:3752), nanoparticle probe technology (See, Elghanian, et al. (1997) Science 277:1078-1081.) and Amplifluor probe technology (See, U.S. Pat. Nos. 5,866,366; 6,090,592; 6,117,635; and 6,117,986).

[0121] In preferred embodiments, molecular beacons are used for detection of the target nucleic acids. Molecular beacons are single stranded oligonucleotides that, unless bound to target, exist in a hairpin conformation. The 5' end of the oligonucleotide contains a fluorescent dye. A quencher dye is attached to the 3' end of the oligonucleotide. When the beacon is not bound to target, the hairpin structure positions the fluorophore and quencher in close proximity, such that no fluorescence can be observed. Once the beacon hybridizes with target, however, the hairpin structure is disrupted, thereby separating the fluorophore and quencher and enabling detection of fluorescence. (See, Kramer F R., 1996, Nat Biotechnol 3:303-8.). Other detection methods include target gene nucleic acids detection via immunological methods, solid phase hybridization methods on filters, chips or any other solid support. In these systems, the hybridization can be monitored by any suitable method known to those skilled in the art, including fluorescence, chemiluminescence, potentiometry, mass spectrometry, plasmon resonance, polarimetry, colorimetry, flow cytometry or scanometry. Nucleotide sequencing, including sequencing by dideoxy termination or sequencing by hybridization (e.g. sequencing using a DNA chip) represents another method to detect and characterize target nucleic acids, such as nuc or MREJ nucleic acid sequences.

Methods

[0122] In preferred embodiments, methods to detect a S. aureus strain in a sample include the step of providing a primer pair, with a first and a second primer. The first and the second primer can anneal under stringent conditions to at least one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or the complements thereof, such as primers that comprise, consist essentially of, or consist of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In preferred embodiments, the primer pairs comprise first and second primers that anneal under stringent conditions to SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8, or the complements thereof. For example, in preferred embodiments, the primer pairs can comprise, consist essentially of, SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8. The sample can be contacted with and allowed to anneal to the primer pair. Preferably, an amplification reaction (e.g., PCR) is performed with the annealed primer pair to amplify S. aureus-specific nuc sequences using the techniques described herein. Amplification products can then be detected using any of the methods described herein.

[0123] In some embodiments, the primer pair includes a first primer that anneals under stringent conditions to SEQ ID NO: 1 or the complement thereof, and a second primer that anneals under stringent conditions to SEQ ID NO: 6, or the complement thereof, such as a primer pair that comprises, consists essentially of, or consists of SEQ ID NO: 1 and SEQ ID NO: 6. In some embodiments, the primer pair is used to amplify nuc sequences present in the sample. Optionally, a probe that anneals under stringent conditions to SEQ ID NO: 9 or 10 or the complement thereof is also provided, for example, a probe that comprises, consists essentially of, or consist of SEQ ID NO: 9 or 10. In some embodiments, the probe is a molecular beacon probe, and the resulting amplification product can be detected by the probe.

[0124] In other preferred embodiments, a first primer that anneals under stringent conditions to SEQ ID NO: 3 or the complement thereof and a second primer that anneals under stringent conditions to SEQ ID NO: 8, or the complement thereof, are provided, such as a primer pair that comprises, consists essentially of, or consists of SEQ ID NO: 3 and SEQ ID NO: 8. In some embodiments, the primer pair is used to amplify nuc sequences present in the sample. Optionally, a probe that anneals under stringent conditions to SEQ ID NO: 11 or 12 or the complement thereof is also provided for example, a probe that comprises, consists essentially of, or consist of SEQ ID NO: 11 or 12. In some embodiments, the probe is a molecular beacon probe.

[0125] Other aspects of the invention relate to methods and compositions for detecting the presence of S. aureus strains and identifying MRSA strains from a sample in a single assay or reaction. The term "single assay" or "single reaction" is intended to refer to the situation in which steps to detect S. aureus and steps to detect MRSA are performed simultaneously, or at substantially the same time, for example in the same physical enclosure. The skilled artisan will appreciate, however, that steps to detect S. aureus and steps to detect MRSA can also be performed sequentially. In preferred embodiments, S. aureus and MRSA are simultaneously detected, for example in a multiplex PCR reaction.

[0126] Some embodiments involve the steps of contacting the sample with at least one primer and/or probe that anneals under stringent conditions to a species-specific sequence of the nuc gene of S. aureus, and contacting the sample with at least one primer and/or probe that anneals under stringent conditions to a sequence that is specific to MREJ sequences of MRSA strains.

[0127] The MRSA-specific primer(s) and/or probe(s) can anneal under stringent conditions to polymorphic MREJ nucleic acids, including, for example, MREJ types i to xx. The phrase MREJ refers to the mec right extremity junction <<mec right extremity junction>>. MREJ's are approximately 1 kilobase (kb) in length and include sequences from the SCCmec right extremity as well as bacterial chromosomal DNA to the right of the SCCmec integration site (See, Huletsky et al., (2004) J. Clin. Microbiol., 42:1875-1884). Based on the determination of the whole-genome sequences of strain N315 and Mu50, the nomenclature was recently reviewed because SCCmec elements are located downstream (and not upstream) of orfX. Consequently, MREP (Mec Right Extremity Polymorphism) is also referred to as MLEP (Mec Left Extremity Polymorphism). By a similar token, MREJ types can be referred to as MLEJ (mec left extremity junction). (Chongtrakool et al., (2006), Antimicrob. Agents Chemother. 50:1001-1012). Nevertheless, any equivalent way to classify S. aureus and namely MRSA strains will be under the scope of this patent, since sequences will be able to specifically detect S. aureus and to identify those which are resistant to methicillin.

[0128] Non-limiting examples MREJ type i to xx sequences are listed in SEQ ID NOs: 14-88. Accordingly, in some embodiments, in addition to at least one S. aureus-specific nuc primer and/or probe, (e.g., an oligonucleotide that hybridizes under stringent conditions to one of the following SEQ ID NO: 200, the complement thereof or any sequence which differs from SEQ ID NO: 200 by 1 to 20 nucleotides, at least one primer and/or probe that specifically anneals under stringent conditions to at least one MREJ sequence of MREJ types i-xx (e.g., SEQ ID NOs: 14-88) or the complement thereof is provided. Exemplary primers and probes and combinations of primers and probes useful for the detection of MRSA of MREJ types i-xx are found in, for example, International Patent Application PCT/CA02/00824, and in U.S. patent application Ser. No. 11/248,438, hereby expressly incorporated by reference in their entireties. For example, in some embodiments, the at least one MRSA-specific primer and/or probe provided in the method is at least 10 nucleotides in length, and can hybridize under stringent conditions to one of the following SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201 (MREJ types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, (MREJ types xi-xx) or 199, or the complement thereof. For example, the MRSA-specific primers can comprise, consist essentially of, or consist of one of the following SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201 (MREJ types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, (MREJ types xi-xx) or 199. nuc-specific primers and/or probes (e.g., comprising an oligonucleotide that hybridizes under stringent conditions to one of the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or the complement thereof, such as oligonucleotides that comprise, consist essentially of, or consist of one of the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) and MRSA (i.e., MREJ)-specific primers and/or probes (e.g., comprising an oligonucleotide that hybridizes under stringent conditions to SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201 (types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, (types xi-xx) or 199, or the complement thereof) are annealed to the nucleic acids of the sample, and the presence of annealed primers and/or probes, or amplification products produced therefrom, is detected, indicating the presence and/or amount of S. aureus as well as MRSA. For example, in some embodiments, the sample is contacted with at least one primer pair comprising oligonucleotides that hybridize under stringent conditions to SEQ ID NOs: 92 and 82; 92 and 83; 92 and 84; 104 and 86; 104 and 87; 104 and 88; 99 and 89; 99 and 199 (for the detection of MREJ type i); SEQ ID NOs: 92 and 82; 92 and 129; 92 and 130; 93 and 83; and 92 and 84; 99 and 89; 99 and 199 (for the detection of MREJ type ii); SEQ ID NOs: 92 and 136; 92 and 137; 92 and 138; 99 and 202; 99 and 144 (for the detection of MREJ type iii); SEQ ID NOs: 92 and 141; 99 and 105; 99 and 150 (for the detection of MREJ type iv); SEQ ID NOs: 92 and 146; 99 and 196; 99 and 155 (for the detection of MREJ type v); SEQ ID NOs: 92 and 152; 99 and 161; (for the detection of MREJ type vi); SEQ ID NOs: 92 and 153; 92 and 154; 99 and 162; 99 and 163 (for the detection of MREJ type vii); SEQ ID NOs: 92 and 162; 92 and 163; 99 and 170 (for the detection of MREJ type viii); SEQ ID NOs: 92 and 168; 99 and 177 (for the detection of MREJ type ix); SEQ ID NOs: 197 and an oligonucleotide that hybridizes under stringent conditions to orf22 (for the detection of MREJ type x); SEQ ID NOs: 189 and 106; 189 and 99; 189 and 190; 189 and 109 (for the detection of MREJ type xi); SEQ ID NOs: 194 and 106; 194 and 99; 104 and 191; 194 and 109 (for the detection of MREJ type xii); SEQ ID NOs: 177 and 106; 177 and 99; 177 and 190; and 177 and 109 (for the detection of MREJ type xiii); SEQ ID NOs: 177 and 106; 177 and 99; 177 and 193; 177 and 109 (for the detection of MREJ type xiv); SEQ ID NOs: 184 and 106; 108 and 99; 184 and 191; 184 and 191 (for the detection of MREJ type xv); SEQ ID NOs: 89 and 109 (for the detection of MREJ type xvi); SEQ ID NOs: 185 and 106; 185 and 99; 185 and 191; 185 and 109 (for the detection of MREJ type xvii); SEQ ID NOs: 186 and 106; 186 and 99; 186 and 193; 186 and 109 (for the detection of MREJ type xviii); SEQ ID NOs: 187 and 106; 107 and 99; 187 and 913; 187 and 109 (for the detection of MREJ type xix); SEQ ID NOs: 188 and 106; 188 and 99; 188 and 913; and 188 and 109 (for the detection of MREJ type xx), or the complement thereof.

[0129] The most clinically relevant MRSA strains have MREJ types i, ii, iii, iv, v, and vii. Accordingly, preferred methods and compositions relate to the detection of S. aureus and MRSA of MREJ types i-v and vii in a sample. At least one S. aureus-specific nuc-specific primer and/or probe is provided, and primers and/or probes useful for the specific detection of MREJ types i, ii, iii, iv, v and vii are provided. For example, in some embodiments, primers and/or probes that hybridize under stringent conditions to each of the following SEQ ID NOs or the complements thereof are provided: SEQ ID NOs: 99, 199, 144, 150, 155, and 163, such as primers and/or probes that comprise, consist essentially of, or consist of at least one of the following SEQ ID NOs: 99, 199, 144, 150, 155, and 163. Optionally, at least one probe comprising an oligonucleotide that hybridizes under stringent conditions to SEQ ID NOs: 126, 128, 130 and 131 or the complement thereof is provided, for the detection of MREJ sequences of types i, ii, iii, iv, v and vii. For example, at least one primer and/or probe that comprises, consists essentially of, or consists of at least one of the following SEQ ID NOs: 126, 128, 130 and 131, is provided.

[0130] In other preferred embodiments, the at least one primer(s) and/or probe(s) that anneal to MREJ sequences comprises a pair of oligonucleotides that hybridize under stringent conditions to SEQ ID NOs: 99 and 199 (for the detection of type i and type ii MREJ); SEQ ID NOs: 99 and 144 (for the detection of type iii MREJ); SEQ ID NOs: 99 and 150 (for the detection of type iv MREJ); SEQ ID NOs: 99 and 155 (for the detection of type v MREJ); and SEQ ID NOs: 99 and 163 (for the detection of type vii MREJ), or the complement thereof. Optionally oligonucleotides that hybridize under stringent conditions to each of SEQ ID NOs: 99 and 199 (for the detection of type i and type ii MREJ); SEQ ID NOs: 99 and 144 (for the detection of type iii MREJ); SEQ ID NOs: 99 and 150 (for the detection of type iv MREJ); SEQ ID NOs: 99 and 155 (for the detection of type v MREJ); and SEQ ID NOs: 99 and 163 (for the detection of type vii MREJ) are provided. Optionally, the sample is also contacted with a probe comprising an oligonucleotide that hybridizes under stringent conditions to the nucleic acid of SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 or 131, for the detection of MREJ sequences, or SEQ ID NOs: 9, 10, 11, and 12 for the detection of S. aureus nuc, or the complements thereof.

[0131] In preferred embodiments, the nuc-specific primer(s) and/or probe(s) comprise at least one first primer pair that hybridizes under stringent conditions to the following oligonucleotide pairs or the complements thereof: SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8; for the detection of S. aureus in a sample. For example, in some embodiments, the nuc-specific primer(s) and/or probe(s) comprise at least one first primer pair that comprises, consists essentially of, or consists of: SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8. Optionally, in embodiments where the sample is contacted with a first primer pair comprising oligonucleotides that hybridize under stringent conditions to SEQ ID NOs: 1 and 5, or 1 and 6, or the complements thereof, (e.g., oligonucleotides that comprise, consist essentially of, or consist of SEQ ID NOs: 1 and 5, or SEQ ID NOs: 1 and 6), the sample can also be contacted with a probe comprising an oligonucleotide that hybridizes under stringent conditions to SEQ ID NO: 9 (e.g., SEQ ID NO: 10) or the complement thereof. Optionally, in embodiments where the sample is contacted with a first primer pair comprising oligonucleotides that hybridize under stringent conditions to SEQ ID NOs: 3 and 7, or SEQ ID NOs: 3 and 8, or the complements thereof, (e.g., oligonucleotides that comprise, consist essentially of, or consist of SEQ ID NOs: 3 and 7, or SEQ ID NOs: 3 and 8), the sample can also be contacted with a probe comprising an oligonucleotide that hybridizes under stringent conditions SEQ ID NO: 11 (e.g., SEQ ID NO: 12) or the complement thereof. Preferably, the first primer pair comprises oligonucleotides that hybridize under stringent conditions to SEQ ID NOs: 1 and 6; or SEQ ID NOs: 3 and 8 or the complements thereof.

[0132] Optionally, the sample is also contacted with at least one probe comprising an oligonucleotide that hybridizes under stringent conditions to SEQ ID NOs: 9, 10, 11, 12, for the detection of S. aureus nuc sequences, or to SEQ ID NOs: 126, 128, 130 or 131, for the detection of MREJ sequences, or the complement thereof, e.g., at least one probe that comprises, consists essentially of, or consists of SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 or 131.

[0133] The presence and/or amount of annealed probe(s) can be detected, or the amount of an amplification product produced through annealing of the primers to the nucleic acids can be detected, as an indication of the presence and/or amount of S. aureus, and as an indication of the presence and/or amount of MRSA.

Compositions and Kits

[0134] Provided herein are also compositions and kits that comprise, consist essentially of, or consist of oligonucleotides described herein. Preferably, oligonucleotides are between 10 and 45 nucleotides in length. For example, oligonucleotides can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35 or more nucleotides in length. As will be understood by those skilled in the art, the nucleic acids of the embodiments disclosed herein can be single-stranded (coding or antisense), or double-stranded, and may be a DNA (genomic, cDNA, or synthetic) or RNA molecule. Additional coding or non-coding sequences may, but need not, be present within a nucleic acid of the embodiments disclosed herein, and a nucleic acid may, but need not, be linked to other molecules and/or support materials.

[0135] Accordingly, some embodiments comprise, consist essentially of, or consist of, at least one oligonucleotide of between about 10 to about 45 nucleotides, and preferably at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35 nucleotides in length which hybridizes under stringent conditions with any of nucleic acids of the following sequences derived from S. aureus nuc sequences or the complements thereof: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, for example, oligonucleotides that comprise, consist essentially of, or consist of at least one of the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Preferred embodiments comprise, consist essentially of, or consist of a primer pair that hybridizes under stringent conditions with any of the pairs of the following SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8, or the complements thereof, for example primer pairs that comprise, consist essentially of, or consist of the following SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8. In some embodiments, at least one probe comprising an oligonucleotide that hybridizes under stringent conditions SEQ ID NOs: 9 and 11 (e.g., SEQ ID NOs: 10 and 12), or the complement thereof, is provided.

[0136] Other aspects relate to compositions useful for the detection of S. aureus and MRSA in a single reaction. Accordingly, some embodiments comprise, consist essentially of, or consist of, at least one primer and/or probe that is preferably between about 10 to about 45 nucleotides in length, such as an oligonucleotide that is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35 in length that hybridizes to an S. aureus-specific nuc sequence, and at least one primer and/or probe that hybridizes to at least one MREJ sequence of MREJ types i-xx. Some embodiments provide at least two primer pairs, wherein a first primer pair hybridizes under stringent conditions to S. aureus-specific nuc sequences (e.g., SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8) and a second primer pair hybridizes to MREJ sequences (e.g., SEQ ID NOs: 92 and 82; 92 and 83; 92 and 84; 104 and 86; 104 and 87; 104 and 88; 99 and 89; 99 and 199 (for the detection of MREJ type i); SEQ ID NOs: 92 and 82; 92 and 129; 92 and 130; 93 and 83; and 92 and 84; 99 and 89; 99 and 199 (for the detection of MREJ type ii); SEQ ID NOs: 92 and 136; 92 and 137; 92 and 138; 99 and 202; 99 and 144 (for the detection of MREJ type iii); SEQ ID NOs: 92 and 141; 99 and 105; 99 and 150 (for the detection of MREJ type iv); SEQ ID NOs: 92 and 146; 99 and 196; 99 and 155 (for the detection of MREJ type v); SEQ ID NOs: 92 and 152; 99 and 161; (for the detection of MREJ type vi); SEQ ID NOs: 92 and 153; 92 and 154; 99 and 162; 99 and 163 (for the detection of MREJ type vii); SEQ ID NOs: 92 and 162; 92 and 163; 99 and 170 (for the detection of MREJ type viii); SEQ ID NOs: 92 and 168; 99 and 177 (for the detection of MREJ type ix); SEQ ID NOs: 197 and an oligonucleotide that hybridizes under stringent conditions to orf22 (for the detection of MREJ type x); SEQ ID NOs: 189 and 106; 189 and 99; 189 and 190; 189 and 109 (for the detection of MREJ type xi); SEQ ID NOs: 194 and 106; 194 and 99; 104 and 191; 194 and 109 (for the detection of MREJ type xii); SEQ ID NOs: 177 and 106; 177 and 99; 177 and 190; and 177 and 109 (for the detection of MREJ type xiii); SEQ ID NOs: 177 and 106; 177 and 99; 177 and 193; 177 and 109 (for the detection of MREJ type xiv); SEQ ID NOs: 184 and 106; 108 and 99; 184 and 191; 184 and 191 (for the detection of MREJ type xv); SEQ ID NOs: 89 and 109 (for the detection of MREJ type xvi); SEQ ID NOs: 185 and 106; 185 and 99; 185 and 191; 185 and 109 (for the detection of MREJ type xvii); SEQ ID NOs: 186 and 106; 186 and 99; 186 and 193; 186 and 109 (for the detection of MREJ type xviii); SEQ ID NOs: 187 and 106; 107 and 99; 187 and 913; 187 and 109 (for the detection of MREJ type xix); SEQ ID NOs: 188 and 106; 188 and 99; 188 and 913; and 188 and 109 (for the detection of MREJ type xx)). In some embodiments, at least one probe(s) that can hybridize to amplification products produced by an S. aureus-specific nuc primer pair and/or MREJ-specific primer pair described herein is also provided (e.g., SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 or 131).

[0137] Accordingly, some embodiments comprise, consist essentially of, or consist of primer pairs that hybridize under stringent conditions to the nucleic acid sequences of:

[0138] SEQ ID NOs: 1 and 6

[0139] SEQ ID NOs: 99 and 199;

[0140] SEQ ID NOs: 99 and 144;

[0141] SEQ ID NOs: 99 and 150;

[0142] SEQ ID NOs: 99 and 155; and

[0143] SEQ ID NOs: 99 and 163, or the complements thereof.

[0144] Other embodiments comprise, consist essentially of, or consist of a plurality of primer pairs, wherein the primers anneal under stringent conditions to the nucleic acid sequences of:

[0145] SEQ ID NOs: 3 and 8;

[0146] SEQ ID NOs: 99 and 199;

[0147] SEQ ID NOs: 99 and 144;

[0148] SEQ ID NOs: 99 and 150;

[0149] SEQ ID NOs: 99 and 155; and

[0150] SEQ ID NOs: 99 and 163, or the complements thereof.

[0151] Still other aspects relate to kits for the detection and/or quantification of S. aureus, or S. aureus and MRSA. In some embodiments, the kits comprise, consist essentially of, or consist of, at least one oligonucleotide of between about 10 to about 45 nucleotides in length, for example at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35 nucleotides in length, which hybridizes under stringent conditions with any of nucleic acids of the following sequences derived from S. aureus nuc sequences or the complements thereof: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Preferred embodiments provide kits that comprise, consist essentially of, or consist of a primer pair that hybridizes under stringent conditions with any of the pairs of the following SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8, or the complements thereof. In some embodiments, the kit provides at least one probe comprising an oligonucleotide that hybridizes under stringent conditions SEQ ID NOs: 9 and 11 (e.g., SEQ ID NOs: 10 and 12), or the complement thereof.

[0152] Other embodiments provide kits useful for the detection of S. aureus and MRSA together. In some embodiments, the kits comprise, consist essentially of, or consist of, at least one primer and/or probe that is between about 10 to about 45 nucleotides in length, for example, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35 nucleotides in length, that hybridizes to an S. aureus-specific nuc sequence, and at least one primer and/or probe that hybridizes to at least one MREJ sequence of MREJ types i-xx. Some embodiments provide kits, wherein the kits include at least two primer pairs. A first primer pair can hybridize under stringent conditions to S. aureus-specific nuc sequences (e.g., primers that are at least 10 nucleotides in length and can hybridize under stringent conditions to SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8, or the complement thereof) and a second primer pair can hybridize under stringent conditions to MREJ sequences (e.g., primers that are at least 10 nucleotides in length and can hybridize under stringent conditions to SEQ ID NOs: 92 and 82; 92 and 83; 92 and 84; 104 and 86; 104 and 87; 104 and 88; 99 and 89; 99 and 199 (for the detection of MREJ type i); SEQ ID NOs: 92 and 82; 92 and 129; 92 and 130; 93 and 83; and 92 and 84; 99 and 89; 99 and 199 (for the detection of MREJ type ii); SEQ ID NOs: 92 and 136; 92 and 137; 92 and 138; 99 and 202; 99 and 144 (for the detection of MREJ type iii); SEQ ID NOs: 92 and 141; 99 and 105; 99 and 150 (for the detection of MREJ type iv); SEQ ID NOs: 92 and 146; 99 and 196; 99 and 155 (for the detection of MREJ type v); SEQ ID NOs: 92 and 152; 99 and 161; (for the detection of MREJ type vi); SEQ ID NOs: 92 and 153; 92 and 154; 99 and 162; 99 and 163 (for the detection of MREJ type vii); SEQ ID NOs: 92 and 162; 92 and 163; 99 and 170 (for the detection of MREJ type viii); SEQ ID NOs: 92 and 168; 99 and 177 (for the detection of MREJ type ix); SEQ ID NOs: 197 and an oligonucleotide that hybridizes under stringent conditions to orf22 (for the detection of MREJ type x); SEQ ID NOs: 189 and 106; 189 and 99; 189 and 190; 189 and 109 (for the detection of MREJ type xi); SEQ ID NOs: 194 and 106; 194 and 99; 104 and 191; 194 and 109 (for the detection of MREJ type xii); SEQ ID NOs: 177 and 106; 177 and 99; 177 and 190; and 177 and 109 (for the detection of MREJ type xiii); SEQ ID NOs: 177 and 106; 177 and 99; 177 and 193; 177 and 109 (for the detection of MREJ type xiv); SEQ ID NOs: 184 and 106; 108 and 99; 184 and 191; 184 and 191 (for the detection of MREJ type xv); SEQ ID NOs: 89 and 109 (for the detection of MREJ type xvi); SEQ ID NOs: 185 and 106; 185 and 99; 185 and 191; 185 and 109 (for the detection of MREJ type xvii); SEQ ID NOs: 186 and 106; 186 and 99; 186 and 193; 186 and 109 (for the detection of MREJ type xviii); SEQ ID NOs: 187 and 106; 107 and 99; 187 and 913; 187 and 109 (for the detection of MREJ type xix); SEQ ID NOs: 188 and 106; 188 and 99; 188 and 913; and 188 and 109 (for the detection of MREJ type xx) or the complements thereof). In some embodiments, the kits include at least one probe(s) that can hybridize under stringent conditions to amplification products produced by an S. aureus-specific nuc primer pair and/or MREJ-specific primer pair described herein is also provided (e.g., a probe comprising an oligonucleotide that can hybridize under stringent conditions to SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 or 131 or the complement thereof).

[0153] Accordingly, some embodiments provide kits that comprise, consist essentially of, or consist of primer pairs that hybridize under stringent conditions to the nucleic acid sequences of:

[0154] SEQ ID NOs: 1 and 6

[0155] SEQ ID NOs: 99 and 199;

[0156] SEQ ID NOs: 99 and 144;

[0157] SEQ ID NOs: 99 and 150;

[0158] SEQ ID NOs: 99 and 155; and

[0159] SEQ ID NOs: 99 and 163, or the complements thereof.

[0160] Other embodiments provide kits that comprise, consist essentially of, or consist of a plurality of primer pairs, wherein the primers anneal under stringent conditions to the nucleic acid sequences of:

[0161] SEQ ID NOs: 3 and 8;

[0162] SEQ ID NOs: 99 and 199;

[0163] SEQ ID NOs: 99 and 144;

[0164] SEQ ID NOs: 99 and 150;

[0165] SEQ ID NOs: 99 and 155; and

[0166] SEQ ID NOs: 99 and 163, or the complements thereof.

[0167] The diagnostic kits, primers and probes disclosed herein can be used to detect and/or identify S. aureus, as well as detect and/or identify both S. aureus and MRSA of MREJ types i to xx, in both in vitro and/or in situ applications. For example, it is contemplated that the kits may be used in combination with any previously described primers/probes for detecting MRSA of MREJ types i to xx. It is also contemplated that the diagnostic kits, primers and probes disclosed herein can be used alone or in combination with any other assay suitable to detect and/or identify microorganisms, including but not limited to: any assay based on nucleic acids detection, any immunoassay, any enzymatic assay, any biochemical assay, any lysotypic assay, any serological assay, any differential culture medium, any enrichment culture medium, any selective culture medium, any specific assay medium, any identification culture medium, any enumeration culture medium, any cellular stain, any culture on specific cell lines, and any infectivity assay on animals.

[0168] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting.

Example 1

[0169] This example illustrates the utility of various primer pairs, chosen for optimized, specific detection of S. aureus from a sample using PCR. SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 were designed to anneal to S. aureus-specific regions of the nuc gene. PCR reaction mixtures included 0.5 μM of each of the indicated primers, 0.2 mM dNTPs (Roche), 2 mM MgCl₂ (SIGMA), 1 unit FASTSTART® Taq DNA polymerase (Roche), 50 mM Tris (EMD), 10 mM KCl (Laboratoire Mat), and 5 mM (NH₄)₂SO₄ (SIGMA).

[0170] For each primer pair tested, three replicates containing varying amounts of chromosomal template DNA were run. One set of reactions included 15 copies of chromosomal template DNA from S. aureus strain ATCC 43300 (MRSA). Another set of reactions included 185 copies of ATCC 43300 template DNA. A negative control was also run, which did not have any added template DNA. Parallel sets of reactions were run with chromosomal template DNA from S. aureus strain ATCC 25923 (MSSA).

[0171] PCR reactions were performed using a SMARTCYCLER® QT-PCR machine (Cepheid). The cycling parameters were as follows: 95° C. for 900 min, followed by 45 cycles of 95° C. for 5 sec, 59° C. for 15 sec and 72° C. for 20 sec. Amplified products were visualized on agarose gels (FIGS. 1A and 1B).

[0172] As shown in FIGS. 1A and 1B, the following primer pairs showed particularly good results in the specific amplification of DNA from both MSSA and MRSA S. aureus strains:

[0173] SEQ ID NOs: 1 and 5

[0174] SEQ ID NOs: 1 and 6

[0175] SEQ ID NOs: 2 and 6

[0176] SEQ ID NOs: 4 and 7

[0177] SEQ ID NOs: 4 and 8

[0178] SEQ ID NOs: 3 and 7, and

[0179] SEQ ID NOs: 3 and 8.

[0180] The primer pair SEQ ID NOs: 2 and 5 was less sensitive, as indicated by the relative amount of amplification product produced, compared to other primer pairs.

Example 2

[0181] The ability to detect S. aureus and to identify MRSA in a single reaction was tested. A multiplex PCR reaction was designed to include primers that anneal under standard PCR conditions to the S. aureus species-specific orfX sequence and a sequence of SCCmec right extremity junction (MREJ) of the most commonly clinically encountered MRSA types (i.e., MRSA of MREJ types i, ii, iii, iv, v, vii). SEQ ID NOs: 99, 199, 144, 150, 155 and 163 were used for the detection of MRSA of MREJ types i, ii, iii, iv, v, and vii. Primers that anneal to S. aureus specific regions of the nuc gene under the same conditions (SEQ ID NOs: 3 and 8) were used in the reaction for the detection of both MRSA and MSSA strains in the test reactions. Molecular beacon probes which are detectable on the SMARTCYCLER® apparatus at FAM, Texas Red and TET channels were designed for hybridization to amplification products of the MRSA specific reactions (SEQ ID NOs: 126 and 130), nuc/S. aureus specific reactions (SEQ ID NO: 12), and an internal control, respectively.

[0182] PCR reactions included 0.9 μM SEQ ID NO: 99, 0.4 μM SEQ ID NO: 199; 0.6 μM SEQ ID NO: 144, 0.3 μM SEQ ID NO: 150, 0.2 μM SEQ ID NO: 155, 0.7 μM SEQ ID NO: 163, 0.1 μM SEQ ID NO: 3, 0.1 μM SEQ ID NO: 8, 0.1 μM SEQ ID NO: 126, 0.1 μM SEQ ID NO: 130, 0.25 μM SEQ ID NO: 12, 0.2 μM control DNA, 0.3 mM dNTPs (Roche) 4 mM MgCl₂ (SIGMA), 2.8 units FASTSTART® Taq DNA polymerase (Roche), 100 mM Tris, pH 8.3 (EMD), 10 mM KCl (Laboratoire Mat), 5 mM (NH4)₂SO₄ (SIGMA), 0.15 mg/mL BSA (SIGMA), 4% Trehalose (SIGMA), 3000 copies of internal control template DNA, 2780 copies of S. epidermidis chromosomal DNA, and either 0, 2.5, 5, 10, 15 or 20 copies MSSA chromosomal DNA (isolated from ATCC strain 25923) or 0, 2.5, 5, 10 or 20 copies of MRSA chromosomal DNA (isolated from ATCC strain 43300).

[0183] PCR reactions were performed in a SMARTCYCLER® instrument (Cepheid). Cycling parameters were as follows: 95° C. for 900 min, followed by 45 cycles of 95° C. for 5 sec, 59° C. for 15 sec and 72° C. for 20 sec. The fluorescence was continuously measured at the appropriate wavelengths, and is graphically depicted in FIGS. 2A and 2B.

[0184] FIG. 2A depicts the fluorescence readings of reactions containing MSSA template DNA. Under these reaction conditions, 2.5 copies of MSSA DNA were easily detected (Texas Red Channel), demonstrating the utility of SEQ ID NOs: 3, 8 and 12 in multiplex PCR. As expected, positive signals are also present in the TET channel indicating that the internal control worked properly, and that no inhibitors were present in the reactions.

[0185] FIG. 2B depicts the fluorescence readings of reactions containing MRSA template DNA. Under these reaction conditions, 2.5 copies of MRSA DNA were easily detected (FAM channel). This demonstrates the utility of SEQ ID NOs: 99, 199, 150, 155, 144, 126, and 130 in a multiplex PCR that can detect all S. aureus strains, including MRSA. As shown in the Texas-Red channel, the nuc-specific primers and probes (SEQ ID NOs: 3, 8, and 12) detected 2.5 copies of DNA. Positive signals are also present in the TET channel, indicating that the internal control worked properly, and that no inhibitors were present in the reactions.

[0186] This example highlights the very high sensitivity obtainable with a PCR multiplex assay that amplifies MREJ sequences from MRSA and nuc sequences from S. aureus with an internal control.

Example 3

[0187] The specificity of a multiplex PCR assay that amplifies MREJ sequences from MRSA and the nuc sequence from S. aureus was analyzed. Chromosomal DNA from 80 bacterial species other than S. aureus was used as template DNA in a multiplex PCR assay as described in Example 2. The strains tested are enumerated in Table 2.

[0188] 1 ng of chromosomal DNA isolated from each species indicated in Table 1 was used in a separate reaction containing 0.9 μM SEQ ID NO: 99, 0.4 μM SEQ ID NO: 199; 0.6 μM SEQ ID NO: 144, 0.3 μM SEQ ID NO: 150, 0.2 μM SEQ ID NO: 155, 0.7 μM SEQ ID NO: 163, 0.1 μM SEQ ID NO: 3, 0.1 μM SEQ ID NO: 8, 0.1 μM SEQ ID NO: 126, 0.1 μM SEQ ID NO: 131, 0.25 μM SEQ ID NO: 11, 0.2 μM internal control DNA, 0.3 mM dNTPs (Roche) 4 mM MgCl₂ (SIGMA), 2.8 units FASTSTART® Taq DNA polymerase (Roche), 100 mM Tris, pH 8.3 (EMD), 10 mM KCl (Laboratoire Mat), 5 mM (NH₄)₂SO₄ (SIGMA), 0.15 mg/mL BSA (SIGMA), 4% Trehalose (SIGMA), 3000 copies of internal control template DNA, and 2780 copies of S. epidermidis DNA.

[0189] Each reaction was performed in triplicate. The reactions were allowed to proceed following the parameters set forth in Example 2. Table 2 summarizes the results of the reactions. No positive signal was observed in the FAM and Texas Red channels for the 80 different species tested. A positive signal was detected in the TET channel for each of the 80 different species tested, indicating that the reactions did not contain inhibitors. The algorithm of interpretation of results is summarized in Table 3.

TABLE-US-00001 TABLE 2 PCR results Strain MRSA IC S. aureus Species number (FAM) (TET) (Texas Red) Acinetobacter baumannii ATCC 19606 - + - Acinetobacter lwoffi CDCF 3697 - + - Actinomyces israelii ATCC 12102 - + - Actinomyces pyogenes ATCC 19411 - + - Bacillus cereus ATCC 14579 - + - Bacteroides fragilis ATCC 25285 - + - Bifidobacterium breve ATCC 15700 - + - Bordetella pertusis ATCC 9797 - + - Corynebacterium genitalium LSPQ3583 - + - Corynebacterium aquaticus ATCC 14665 - + - Corynebacterium bovis ATCC 7715 - + - Corynebacterium flavescens ATCC 10340 - + - Enterobacter cloacae ATCC 13047 - + - Enterococcus faecalis ATCC19433 - + - Enterococcus faecium ATCC 19434 - + - Enterococcus flavescens ATCC 49996 - + - Enterrococcus gallinarum ATCC 49573 - + - Enterrococcus hirae ATCC 8043 - + - Escherichia coli ATCC 23511 - + - Helicobacter pylori IDI-2019 - + - Fusobacterium nucleatum subsp. ATCC 10953 - + - Polymorphum Gardnerella vaginalis ATCC 14019 - + - Haemophilus influenzae ATCC 9006 - + - Homo sapiens 2.16 - + - Klebsiella pneumoniae ATCC 13883 - + - Lactobacillus crispatus ATCC 33820 - + - Listeria monocytogenes L 374 - + - Micrococcus luteus ATCC 9341 - + - Moraxella catarrhalis ATCC 43628 - + - Neisseria gonorrhoeae ATCC 35201 - + - Neisseria meningitides ATCC 13077 - + - Pasteurella aerogenes ATCC 27883 - + - Peptostreptococcus anaerobius ATCC 27337 - + - Peptostreptococcus asaccharolyticus LSPQ 2639 - + - Porphyromonas asaccharolytica ATCC 25260 - + - Prevotella melaninogenica ATCC 25845 - + - Propionibacterium acnes ATCC 6919 - + - Proteus mirabilis ATCC 29906 - + - Pseudomonas aeruginosa ATCC 35554 - + - Pseudomonas fluorescens ATCC 13525 - + - Salmonella typhimurium ATCC 14028 - + - Serratia marcescens ATCC 13880 - + - Shigella sonnei ATCC 29930 - + - Staphylococcus arlettae CCRI-9265 - + - Staphylococcus auricularis R413 - + - Staphylococcus capitis CCRI-9572 - + - Staphylococcus caprae CCRI-9117 - + - Staphylococcus carnosus R714 - + - Staphylococcus chromogenes ATCC 43764 - + - Staphylococcus cohnii subsp. R570 - + - Urealyticum Staphylococcus delphini ATCC 49171 - + - Staphylococcus epidermidis ATCC 35984 - + - Staphylococcus equorum ATCC 43958 - + - Staphylococcus felis ATCC 49168 - + - Staphylococcus gallinarum ATCC 35539 - + - Staphylococcus haemolyticus ATCC 29970 - + - Staphylococcus hominis CCRI-1347 - + - Staphylococcus intermedius ATCC 29663 - + - Staphylococcus kloosii ATCC 43959 - + - Staphylococcus lentus ATCC 29070 - + - Staphylococcus lugdunensis ATCC 43809 - + - Staphylococcus pasteuri ATCC 51129 - + - Staphylococcus pulvereri ATCC 51698 - + - Staphylococcus saprophyticus ATCC 15305 - + - Staphylococcus sciuri R573 - + - Staphylococcus simulans ATCC 27848 - + - Staphylococcus warneri ATCC 35985 - + - Staphylococcus xylosus LSPQ2517 - + - Streptococcus agalactiae ATCC 12973 - + - Streptococcus anginosus ATCC 33397 - + - Streptococcus mitis ATCC 49456 - + - Streptococcus mutans ATCC 25175 - + - Streptococcus pneumoniae ATCC 49619 - + - Streptococcus pyogenes ATCC 12384 - + - Streptococcus salivarius ATCC 7073 - + - Streptococcus sanguinis ATCC 10556 - + - Streptococcus suis ATCC 43765 - + - Yersinia enterocolitica ATCC 23715 - + - Candida albicans ATCC 10231 - + - Candida glabrata ATCC 66032 - + -

TABLE-US-00002 TABLE 3 FAM Assay Texas-Red IC (TET) Result Assay Result Result Reported Reported Reported Interpretation of Result Negative Negative PASS No S. aureus DNA detected Positive Positive or N/A MRSA DNA detected Negative Negative Positive N/A S. aureus DNA detected, no MRSA DNA detected Unresolved Fail Unresolved- inhibitory specimen or reagent failure

[0190] This example highlights the complete specificity reached with a PCR multiplex assay that amplifies MREJ sequences from MRSA and nuc sequence from S. aureus with an internal control.

Example 4

[0191] The ability of a multiplex PCR assay that amplifies MREJ sequences from MRSA and nuc sequence from S. aureus to accurately detect S. aureus and identify MRSA directly from wound specimens was tested.

[0192] A multiplex PCR reaction was designed to include primers to amplify sequences specific to the MREJ regions of the most clinically relevant MRSA (e.g., primers that anneal to S. aureus species-specific orfX sequences and SCCmec sequences), as well as primers that anneal to S. aureus specific regions of the nuc gene of all S. aureus strains (e.g., MRSA and MSSA), under the same conditions. Briefly, SEQ ID NOs: 99, 199, 144, 150, 155 and 163 were used for amplification of sequences of the MREJ region of various MRSA of MREJ types i, ii, iii, iv, v, and vii. Primers that anneal to S. aureus specific regions of the nuc gene under the same conditions (SEQ ID NOs: 3 and 8) were used in the reaction for the detection of both MRSA and MSSA strains in the test reactions. Molecular beacon probes which are detectable on the SMARTCYCLER® apparatus at FAM, Texas Red and Tet channels were designed for hybridization to amplification products of the MRSA specific reactions (SEQ ID NOs: 126 and 130), nuc/S. aureus specific reactions (SEQ ID NO: 12), and the internal control, respectively.

[0193] One hundred and three wound samples were collected on patients using Amies liquid swabs (Copan Diagnostics, Inc). Samples were cultured and subcultured on blood agar plates (Becton Dickinson). Based on their morphology, suspected S. aureus were identified with a coagulase test (Jorgenson, J. H., and W. E. Kloos. 1987. Staphylococcal Infections, in B. B. Wentworth (ed.), Diagnostic procedures for bacterial infections, 7th ed., American Public Health Association, Washington, D.C.) and in some cases with latex agglutination (Staphaurex, Remel Inc.) Methicillin resistance was determined using the VITEK® bacterial identification system (bioMerieux, Durham, N.C.).

[0194] DNA was isolated from the isolates using the IDI® lysis kit (GeneOhm Sciences, Inc.). A swab of the isolate was broken in 1 mL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and vortexed for 1 min at high speed. 50 μL of the cell suspensions were transferred to a lysis tube containing glass beads and vortexed for 5 minutes at high speed. The tubes were centrifuged at 13,000 rpms for 2 min and heated at 95° C. for 2 minutes. The tube was placed on ice until used in the reaction.

[0195] 3 μL of the lysis reaction was added to a PCR mix that contained 0.9 μM SEQ ID NO: 99, 0.4 μM SEQ ID NO: 199, 0.6 μM SEQ ID NO: 144, 0.3 μM SEQ ID NO: 150, 0.2 μM SEQ ID NO: 155, 0.7 μM SEQ ID NO: 163, 0.1 μM SEQ ID NO: 3, 0.1 μM SEQ ID NO: 8, 0.1 μM SEQ ID NO: 126, 0.1 μM SEQ ID NO: 130, 0.25 μM SEQ ID NO:12, 0.2 μM internal control DNA, 0.3 μM dNTPs (Roche), 4 mM MgCl₂ (SIGMA), 2.8 units FASTSTART® Taq polymerase (Roche), 100 mM Tris, pH 8.3 (EMD), 10 mM KCl (LaboratoireMat), 5 mM (NH₄)₂SO₄ (SIGMA), 0.15 mg/mL BSA (SIGMA) 4% trehalose (SIGMA), 3000 copies internal control DNA, and 2780 copies S. epidermidis chromosomal DNA.

[0196] PCR was carried out in a SMARTCYCLER® (Cepheid) using the same cycling parameters as described in Example 2. For each specimen, the cycle threshold (CT) in FAM, Texas-Red, and TET channels was determined using the SMARTCYCLER® software. Assay results were interpreted as indicated in Table 3:

[0197] The multiplex PCR assay above is designed such that any S. aureus strain produces a positive signal in the Texas-Red channel. The presence of a clinically relevant MRSA will produce a positive signal in the FAM channel. Accordingly, a negative result in the FAM channel combined with a positive result in the Texas-Red channel is indicative of the presence of MSSA.

[0198] In instances where a discordant result appeared between culture assays described above, and the multiplex PCR reaction, Tryptic Soy Broth was added to the TE buffer tube containing the swab, and incubated overnight, at 35° C. 50 μL of the overnight culture was plated on blood agar plates and isolates were identified as MRSA, MSSA, or negative (no S. aureus).

[0199] The data collected are depicted in Tables 4A, 4B and 4C, below.

TABLE-US-00003 TABLE 4A (A) PCR Un- MRSA MSSA Negative Total resolved Culture MRSA 27 (32)* 0 (0) 0 (0) 27 (32) 1 (0) MSSA 2 (2) 18 (19) 1 (0) 21 (21) 1 (0) Negative 4 (0) 1 (1) 43 (45) 48 (46) 5 (4) Total 33 (34) 19 (20) 44 (45) 96 (99) 7 (4) *before resolution of discordant results (after resolution of discordant results)

TABLE-US-00004 TABLE 4B before resolution after resolution MRSA sensitivity 100% (27/27) 100% (32/32) MSSA sensitivity 85.7% (18/21) 90.5% (19/21) S. aureus sensitivity 97.9% (47/48) 100% (53/53) Specificity 89.6% (43/48) 97.8% (45/46) Unresolved 6.8% (7/103) 3.9% (4/103)

TABLE-US-00005 TABLE 4C ##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005##

[0200] As shown in Tables 4A and 4B, the multiplex assay is 100% sensitive for MRSA, indicating that every positive MRSA result achieved in the PCR assay corresponded to a positive result in the culture identification, both before and after resolution. The sensitivity of the PCR assay for S. aureus detection after resolution was 90.5%, with 19 of 21 of MSSA strains showing a positive result in the PCR assay. Importantly, however, the two strains that were incorrectly identified as not being MSSA are strains that were formerly MSSA but lost a portion of the SCCmec element and retained the junction near orfX to which the PCR amplification primers hybridize.

[0201] Table 4C shows the individual PCR and culture results for each of the 103 wound specimens following the resolution of discordant results. The shaded entries indicate that the results obtained in the culture test and in the PCR assay were in agreement. The column labeled (CT) indicates the PCR cycle in which a positive signal becomes detectable over the background noise. As shown in the table, four samples were not able to be resolved in the PCR assay, due to the presence of reaction inhibitors in the sample.

[0202] The results above demonstrate the high sensitivity and specificity of the multiplex PCR assay applied directly to wound specimens. Accordingly, the multiplex assay offers the first convenient, reliable, sensitive, and specific assay specific for both MRSA and MSSA.

[0203] The methods, compositions, and devices described herein are presently representative of preferred embodiments, they are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the disclosure. Accordingly, it will be apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0204] As used in the claims below and throughout this disclosure, by the phrase "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0205] Numerous literature and patent references have been cited in the present patent application. Each and every reference that is cited in this patent application is hereby expressly incorporated by reference in its entirety.

Sequence CWU 1

1

205124DNAArtificial SequenceStaphylococcus aureus 1acagaatact tattaagtgc tggc 24227DNAArtificial SequenceStaphylococcus aureus 2gtttcaatat tacttatagg gatggct 27324DNAArtificial SequenceStaphylococcus aureus 3ctgatggaaa aatggtaaac gaag 24422DNAArtificial SequenceStaphylococcus aureus 4atggaaaaat ggtaaacgaa gc 22522DNAArtificial SequenceStaphylococcus aureus 5tgctgagcta cttagacttg aa 22622DNAArtificial SequenceStaphylococcus aureus 6gcatttgctg agctacttag ac 22725DNAArtificial SequenceStaphylococcus aureus 7gttgttcatg tgtattgtta ggttt 25822DNAArtificial SequenceStaphylococcus aureus 8ttattgacct gaatcagcgt tg 22930DNAArtificial SequenceStaphylococcus aureus 9cgaaacatta ctgatagcca tccctataag 301040DNAArtificial SequenceStaphylococcus aureus 10cgcaccgaaa cattactgat agccatccct ataaggtgcg 401129DNAArtificial SequenceStaphylococcus aureus 11acataagcaa ctttagccaa gccttgacg 291241DNAArtificial SequenceStaphylococcus aureus 12cgcgcaacat aagcaacttt agccaagcct tgacgtgcgc g 411326DNAArtificial SequenceStaphylococcus aureus 13atgtcaaaaa tcatgaacct cattac 26143050DNAArtificial SequenceStaphylococcus aureus 14tcgtgccatt gatgcagagg gacatacatt agatatttgg ttgcgtaagc aacgagataa 60tcattcagca tatgcgttta tcaaacgtct cattaaacaa tttggtaaac ctcaaaaggt 120aattacagat caggcacctt caacgaaggt agcaatggct aaagtaatta aagcttttaa 180acttaaacct gactgtcatt gtacatcgaa atatctgaat aacctcattg agcaagatca 240ccgtcatatt aaagtaagaa agacaaggta tcaaagtatc aatacagcaa agaatacttt 300aaaaggtatt gaatgtattt acgctctata taaaaagaac cgcaggtctc ttcagatcta 360cggattttcg ccatgccacg aaattagcat catgctagca agttaagcga acactgacat 420gataaattag tggttagcta tattttttta ctttgcaaca gaaccgaaaa taatctcttc 480aatttatttt tatatgaatc ctgtgactca atgattgtaa tatctaaaga tttcagttca 540tcatagacaa tgttcttttc aacatttttt atagcaaatt gattaaataa attctctaat 600ttctcccgtt tgatttcact accatagatt atattatcat tgatatagtc aatgaataat 660gacaaattat cactcataac agtcccaacc cctttatttt gatagactaa ttatcttcat 720cattgtaaaa caaattacac cctttaaatt taactcaact taaatatcga caaattaaaa 780aacaataaaa ttacttgaat attattcata atatattaac aactttatta tactgctctt 840tatatataaa atcattaata attaaacaag ccttaaaata tttaactttt ttgtgattat 900tacacattat cttatctgct ctttatcacc ataaaaatag aaaaaacaag attcctaaag 960aatataggaa tcttgtttca gactgtggac aaactgattt tttatcagtt agcttattta 1020gaaagtttta tttaaattac agtttctatt tttattagat cacaatttta ttttagctct 1080tgttcaagta atcatttttc gccaaaaact ttatactgaa tagcttctac attaaatact 1140ttgtcaatga gatcatctac atctttaaat tcagaataat ttgcatatgg atctataaaa 1200taaaattgtg gttctttacc ggaaacatta aatattctta atattaaata tttctgctta 1260tattctttca tagcaaacat ttcatttagc gacataaaaa atggttcctc aatactagaa 1320gatgtagatg ttttaatttc aataaatttt tctacagctt tatctgtatt tgttggatca 1380aaagctacta aatcatagcc atgaccgtgt tgagagcctg gattatcatt taaaatattc 1440ctaaactgtt ctttcttatc ttcgtctatt ttattatcaa ttagctcatt aaagtaattt 1500agcgctaatt tttctccaac tttaccggtt aatttattct ctttatttga tttttcaatt 1560tctgaatcat ttttagtagt ctttgataca ccttttttat attttggaat tattccttta 1620ggtgcttcca cttccttgag tgtcttatct ttttgtgctg ttctaatttc ttcaatttcg 1680ctgtcttcct gtatttcgtc tatgctattg accaagctat cataggatgt ttttgtaact 1740tttgaagcta attcattaaa tagttctaaa aatttcttta aatcctctag catatcttct 1800tctgtgaatc cttcattcaa atcataatat ttgaatctta ttgatccatg agaatatcct 1860gatggataat cattttttaa atcataagat gaatctttat tttctgcgta ataaaatctt 1920ccagtattaa attcatttga tgtaatatat ttattgagtt cggaagataa agttaatgct 1980ctttgttttg cagcattttt atcccgcgga aacatatcac ttatctttga ccatccttga 2040ttcaaagata agtatatgcc ttctccttcc ggatgaaaaa gatataccaa ataatatcca 2100tcctttgttt cttttgttat attctcatca tatattgaaa tccaaggaac tttactatag 2160ttcccagtag caaccttccc tacaactgaa tatttatctt cttttatatg cacttttaac 2220tgcttgggta acttatcatg gactaaagtt ttatatagat cacctttatc ccaatcagat 2280tttttaacta cattattggt acgtttctct ttaattaatt taaggacctg cataaagttg 2340tctatcattt gaaattccct cctattataa aatatattat gtctcatttt cttcaatatg 2400tacttattta tattttaccg taatttacta tatttagttg cagaaagaat tttctcaaag 2460ctagaacttt gcttcactat aagtattcag tataaagaat atttcgctat tatttacttg 2520aaatgaaaga ctgcggaggc taactatgtc aaaaatcatg aacctcatta cttatgataa 2580gcttctcctc gcataatctt aaatgctctg tacacttgtt caattaacac aacccgcatc 2640atttgatgtg ggaatgtcat tttgctgaat gatagtgcgt agttactgcg ttgtaagacg 2700tccttgtgca ggccgtttga tccgccaatg acgaaaacaa agtcgctttg cccttgggtc 2760atgcgttggt tcaattcttg ggccaatcct tcggaagata gcatctttcc ttgtatttct 2820aatgtaatga ctgtggattg tggtttgatt ttggctagta ttcgttggcc ttctttttct 2880tttacttgct caatttcttt gtcactcata ttttctggtg ctttttcgtc tggaacttct 2940atgatgtcta tcttggtgta tgggcctaaa cgtttttcat attctgctat ggcttgcttc 3000caatatttct cttttagttt ccctacagct aaaatggtga ttttcatgtc 305015385DNAArtificial SequenceStaphylococcus aureus 15ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgagcaa 120gtgtatagag catttaagat tatgcgtgga gaagcttatc ataagtaatg aggttcatga 180tttttgacat agttagcctc cgcagtcttt catttcaagt aaataatagc gaaatattct 240ttatactgaa tacttatagt gaagcaaagt tctagctttg agaaaattct ttctgcaact 300aaatatagta aattacggta aaatataaat aagtacatat tgaagaaaat gagacataat 360atattttata ataggaggga atttc 38516385DNAArtificial SequenceStaphylococcus aureus 16ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgagcaa 120gtgtatagag catttaagat tatgcgtgga gaagcttatc ataagtaatg aggttcatga 180tttttgacat agttagcctc cgcagtcttt catttcaagt aaataatagc gaaatattct 240ttatactgaa tacttatagt gaagcaaagt tctagctttg agaaaattct ttctgcaact 300aaatatagta aattacggta aaatataaat aagtacatat tgaagaaaat gagacataat 360atattttata ataggaggga atttc 38517385DNAArtificial SequenceStaphylococcus aureus 17ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgagcaa 120gtgtatagag catttaagat tatgcgtgga gaagcttatc ataagtaatg aggttcatga 180tttttgacat agttagcctc cgcagtcttt catttcaagt aaataatagc gaaatattct 240ttatactgaa tacttatagt gaagcaaagt tctagctttg agaaaattct ttctgcaact 300aaatatagta aattacggta aaatataaat aagtacatat tgaagaaaat gagacataat 360atattttata ataggaggga atttc 38518385DNAArtificial SequenceStaphylococcus aureus 18ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgcg 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacaaag catttaagat tatgcgagga gaagcttatc ataagtaatg aggttcatga 180tttttgacat agttagcctc cgcagtcttt catttcaagt aaataatagc gaaatattct 240ttatactgaa tacttatagt gaagcaaagt tctagctttg agaaaattct ttctgcaact 300aaatatagta aattacggta aaatataaat aagtacatat tgaagaaaat gagacataat 360atattttata ataggaggga atttc 38519340DNAArtificial SequenceStaphylococcus aureus 19cgcagtaact acgcgctatc attcagcaaa atgacattcc cacatcaaat gatgcgggtt 60gtgttagttg agcaagtgta catagcattt aagattatgc gaggagaagc ttatcataag 120taatgaggtt catgattttt gacatagtta gcctccgcag tctttcattt caagtaaata 180atagcgaaat attctttata ctgaatactt atagtgaagc aaagttctag ctttgagaaa 240attctttctg caactaaata tagtaaatta cggtaaaata taaataagta catattgaag 300aaaatgagac ataatatatt ttataatagg agggaatttc 34020369DNAArtificial SequenceStaphylococcus aureus 20caaacggcct gcacaaggac gtcttacaac gcagtaacta cgcactatca ttcagcaaaa 60tgacattccc acatcaaatg atgcgggttg tgttaattga acaagtgtac agagcattta 120agattatgcg aggagaagct tatcataagt aatgaggttc atgatttttg acatagttag 180cctccgcagt ctttcatttc aagtaaataa tagcgaaata ttctttatac tgaatactta 240tagtgaagca aagttctagc tttgagaaaa ttctttctgc aactaaatat agtaaattac 300ggtaaaatat aaataagtac atattgaaga aaatgagaca taatatattt tataatagga 360gggaatttc 369212480DNAArtificial SequenceStaphylococcus aureus 21aaaccgtctg gcaaacgaat taatgctatt caaattttaa ataaagagac aggtaagttt 60gaaaatattg atttaaaacg tgtatatcac gtaacgatga atgacttcac agcatcaggt 120ggcgacggat atagtatgtt cggtggtcct agagaagaag gtatttcatt agatcaagta 180ctagcaagtt atttaaaaac agctaactta gctaagtatg atacgacaga accacaacgt 240atgttattag gtaaaccagc agtaagtgaa caaccagcta aaggacaaca aggtagcaaa 300ggtagtaagt ctggtaaaga tacacaacca attggtgacg acaaagtgat ggatccagcg 360aaaaaaccag ctccaggtaa agttgtattg ttgctagcgc atagaggaac tgttagtagc 420ggtacagaag gttctggtcg cacaatagaa ggagctactg tatcaagcaa gagtgggaaa 480caattggcta gaatgtcagt gcctaaaggt agcgcgcatg agaaacagtt accaaaaact 540ggaactaatc aaagttcaag cccagaagcg atgtttgtat tattagcagg tataggttta 600atcgcgactg tacgacgtag aaaagctagc taaaatatat tgaaaataat actactgtat 660ttcttaaata agaggtacgg tagtgttttt ttatgaaaaa aagcgataac cgttgataaa 720tatgggatat aaaaacgagg ataagtaata agacatcaag gtgtttatcc acagaaatgg 780ggatagttat ccagaattgt gtacaattta aagagaaata cccacaatgc ccacagagtt 840acccacaaat acacaggtta tacactaaaa atcgggcata aatgtcagga aaatatcaaa 900aactgcaaaa aatattggta taataagagg gaacagtgtg aacaagttaa taacttgtgg 960ataactggaa agttgataac aatttggagg accaaacgac atgaaaatca ccattttagc 1020tgtagggaaa ctaaaagaga aatattggaa gcaagccata gcagaatatg aaaaacgttt 1080aggcccatac accaagatag acatcataga agttccagac gaaaaagcac cagaaaatat 1140gagtgacaaa gaaattgagc aagtaaaaga aaaagaaggc caacgaatac tagccaaaat 1200caaaccacaa tccacagtca ttacattaga aatacaagga aagatgctat cttccgaagg 1260attggcccaa gaattgaacc aacgcatgac ccaagggcaa agcgactttg ttttcgtcat 1320tggcggatca aacggcctgc acaaggacgt cttacaacgc agtaactacg cactatcatt 1380cagcaaaatg acattcccac atcaaatgat gcgggttgtg ttaattgaac aagtgtacag 1440agcatttaag attatgcgag gagaagctta tcataagtaa tgaggttcat gatttttgac 1500atagttagcc tccgcagtct ttcatttcaa gtaaataata gcgaaatatt ctttatactg 1560aatacttata gtgaagcaaa gttctagctt tgagaaaatt ctttctgcaa ctaaatatag 1620taaattacgg taaaatataa ataagtacat attgaagaaa atgagacata atatatttta 1680taataggagg gaatttcaaa tgatagacaa ctttatgcag gtccttaaat taattaaaga 1740gaaacgtacc aataatgtag ttaaaaaatc tgattgggat aaaggtgatc tatataaaac 1800tttagtccat gataagttac ccaagcagtt aaaagtgcat ataaaagaag ataaatattc 1860agttgtaggg aaggttgcta ctgggaacta tagtaaagtt ccttggattt caatatatga 1920tgagaatata acaaaagaaa caaaggatgg atattatttg gtatatcttt ttcatccgga 1980aggagaaggc atatacttat ctttgaatca aggatggtca aagataagtg atatgtttcc 2040gcgggataaa aatgctgcaa aacaaagagc attaacttta tcttccgaac tcaataaata 2100tattacatca aatgaattta atactggaag attttattac gcagaaaata aagattcatc 2160ttatgattta aaaaatgatt atccatcagg atattctcat ggatcaataa gattcaaata 2220ttatgatttg aatgaaggat tcacagaaga agatatgcta gaggatttaa agaaattttt 2280agaactattt aatgaattag cttcaaaagt tacaaaaaca tcctatgata gcttggtcaa 2340tagcatagac gaaatacagg aagacagcga aattgaagaa attagaacag cacaaaaaga 2400taagacactc aaggaagtgg aagcacctaa aggaataatt ccaaaatata aaaaaggtgt 2460atcaaagact actaaaaatg 248022709DNAArtificial SequenceStaphylococcus aureus 22tacattagaa atacaaggaa agatgctatc ttccgaagga ttggcccaag aattgaacca 60acgcatgacc caagggcaaa gcgactttgt tttcgtcatt ggcggatcaa acggcctgca 120caaggacgtc ttacaacgca gtaactacgc actatcattc agcaaaatga cattcccaca 180tcaaatgatg cgggttgtgt taattgaaca agtgtacaga gcatttaaga ttatgcgagg 240agaagcttat cataagtaat gaggttcatg atttttgaca tagttagcct ccgcagtctt 300tcatttcaag taaataatag cgaaatattc tttatactga atacttatag tgaagcaaag 360ttctagcttt gagaaaattc tttctgcaac taaatatagt aaattacggt aaaatataaa 420taagtacata ttgaagaaaa tgagacataa tatattttat aataggaggg aatttcaaat 480gatagacaac tttatgcagg tccttaaatt aattaaagag aaacgtacca ataatgtagt 540taaaaaatct gattgggata aaggtgatct atataaaact ttagtccatg ataagttacc 600caagcagtta aaagtgcata taaaagaaga taaatattca gttgtaggga aggttgctac 660tgggaactat agtaaagttc cttggatttc aatatatgat gagaatata 709233050DNAArtificial SequenceStaphylococcus aureus 23acctcattga gcaagatcac cgtcatatta aagtaagaaa gacaaggtat caaagtatca 60atacagcaaa gaatacttta aaaggtattg aatgtattta cgctctatat aaaaagaacc 120gcaggtctct tcagatctac ggattttcgc catgccacga aattagcatc atgctagcaa 180gttaagcgaa cactgacatg ataaattagt ggttagctat atttttttac tttgcaacag 240aaccgaaaat aatctcttca atttattttt atatgaatcc tgtgactcaa tgattgtaat 300atctaaagat ttcagttcat catagacaat gttcttttca acatttttta tagcaaattg 360attaaataaa ttctctaatt tctcccgttt gatttcacta ccatagatta tattatcatt 420gatatagtca atgaataatg acaaattatc actcataaca gtcccaaccc ctttcttttg 480atagactaat tatcttcatc attgtaaaac aaattacacc ctttaaattt aactcaactt 540aaatatcgac aaattaaaaa acaataaaat tacttgaata ttattcataa tatattaaca 600actttattat actgctcttt atatataaaa tcattaataa ttaaacaagc cttaaaatat 660ttaacttttt tgtgattatt acacattatc ttatctgctc tttatcacca taaaaataga 720aaaaacaaga ttcctaaaga atataggaat cttgtttcag actgtggaca aactgatttt 780ttatcagtta gcttatttag aaagttttat ttaaattaca gtttctattt ttattagatc 840acaattttat tttagctctt gttcaagtaa tcatttttcg ccaaaaactt tatactgaat 900agcttctaca ttaaatactt tgtcaatgag atcatctaca tctttaaatt cagaataatt 960tgcatatgga tctataaaat aaaattgtgg ttctttaccg gaaacattaa atattcttaa 1020tattaaatat ttctgcttat attctttcat agcaaacatt tcatttagcg acataaaaaa 1080tggttcctca atactagaag atgtagatgt tttaatttca ataaattttt ctacagcttt 1140atctgtattt gttggatcaa aagctactaa atcatagcca tgaccgtgtt gagagcctgg 1200attatcattt aaaatattcc taaactgttc tttcttatct tcgtctattt tattatcaat 1260tagctcatta aagtaattta gcgctaattt ttctccaact ttaccggtta atttattctc 1320tttatttgat ttttcaattt ctgaatcatt tttagtagtc tttgatacac cttttttata 1380ttttggaatt attcctttag gtgcttccac ttccttgagt gtcttatctt tttgtgctgt 1440tctaatttct tcaatttcgc tgtcttcctg tatttcgtct atgctattga ccaagctatc 1500ataggatgtt tttgtaactt ttgaagctaa ttcattaaat agttctaaaa atttctttaa 1560atcctctagc atatcttctt ctgtgaatcc ttcattcaaa tcataatatt tgaatcttat 1620tgatccatga gaatatcctg atggataatc attttttaaa tcataagatg aatctttatt 1680ttctgcgtaa taaaatcttc cagtattaaa ttcatttgat gtaatatatt tattgagttc 1740ggaagataaa gttaatgctc tttgttttgc agcattttta tcccgcggaa acatatcact 1800tatctttgac catccttgat tcaaagataa gtatatgcct tctccttccg gatgaaaaag 1860atataccaaa taatatccat cctttgtttc ttttgttata ttctcatcat atattgaaat 1920ccaaggaact ttactatagt tcccagtagc aaccttccct acaactgaat atttatcttc 1980ttttatatgc acttttaact gcttgggtaa cttatcatgg actaaagttt tatatagatc 2040acctttatcc caatcagatt ttttaactac attattggta cgtttctctt taattaattt 2100aaggacctgc ataaagttgt ctatcatttg aaattccctc ctattataaa atatattatg 2160tctcattttc ttcaatatgt acttatttat attttaccgt aatttactat atttagttgc 2220agaaagaatt ttctcaaagc tagaactttg cttcactata agtattcagt ataaagaata 2280tttcgctatt atttacttga aatgaaagac tgcggaggct aactatgtca aaaatcatga 2340acctcattac ttatgataag cttcttaaaa acataacagc aattcacata aacctcatat 2400gttctgatac attcaaaatc cctttatgaa gcggctgaaa aaaccgcatc atttatgata 2460tgcttctcca cgcataatct taaatgctct atacacttgc tcaattaaca caacccgcat 2520catttgatgt gggaatgtca ttttgctgaa tgatagtgcg tagttactgc gttgtaagac 2580gtccttgtgc aggccgtttg atccgccaat gacgaataca aagtcgcttt gcccttgggt 2640catgcgttgg ttcaattctt gggccaatcc ttcggaagat agcatctttc cttgtatttc 2700taatgtaatg actgtggatt gtggtttaat tttggctagt attcgttggc cttctttttc 2760ttttacttgc tcaatttctt tgtcgctcat attttctggt gctttttcgt ctggaacttc 2820tatgatgtct atcttggtgt atgggcctaa acgtttttca tattctgcta tggcttgctt 2880ccaatatttc tcttttagtt tccctacagc taaaatggtg attttcatgt cgtttggtcc 2940tccaaattgt tatcaacttt ccagttatcc acaagttatt aacttgttca cactgttccc 3000tcttattata ccaatatttt ttgcagtttt tgatattttc ctgacattta 305024960DNAArtificial SequenceStaphylococcus aureus 24ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcatatc ataaatgatg cggttttttc 180agccgcttca taaagggatt ttgaatgtat cagaacatat gaggtttatg tgaattgctg 240ttatgttttt aagaagctta tcataagtaa tgaggttcat gatttttgac atagttagcc 300tccgcagtct ttcatttcaa gtaaataata gcgaaatatt ctttatactg aatacttata 360gtgaagcaaa gttctagctt tgagaaaatt ctttctgcaa ctaaatatag taaattacgg 420taaaatataa ataagtacat attgaagaaa atgagacata atatatttta taataggagg 480ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 540ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgagcaa 600gtgtatagag catttaagat tatgcgtgga gaagcatatc ataaatgatg cggttttttc 660agccgcttca taaagggatt ttgaatgtat cagaacatat gaggtttatg tgaattgctg 720ttatgttttt aagaagctta tcataagtaa tgaggttcat gatttttgac atagttagcc 780tccgcagtct ttcatttcaa gtaaataata gcgaaatatt ctttatactg aatacttata 840gtgaagcaaa gttctagctt tgagaaaatt ctttctgcaa ctaaatatag taaattacgg 900taaaatataa ataagtacat attgaagaaa atgagacata atatatttta taataggagg 96025480DNAArtificial SequenceStaphylococcus aureus 25ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgagcaa 120gtgtatagag catttaagat tatgcgtgga gaagcatatc ataaatgatg cggttttttc 180agccgcttca

taaagggatt ttgaatgtat cagaacatat gaggtttatg tgaattgctg 240ttatgttttt aagaagctta tcataagtaa tgaggttcat gatttttgac atagttagcc 300tccgcagtct ttcatttcaa gtaaataata gcgaaatatt ctttatactg aatacttata 360gtgaagcaaa gttctagctt tgagaaaatt ctttctgcaa ctaaatatag taaattacgg 420taaaatataa ataagtacat attgaagaaa atgagacata atatatttta taataggagg 48026458DNAArtificial SequenceStaphylococcus aureus 26ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcatatc ataaatgatg cggttttttc 180agccgcttca taaagggatt ttgaatgtat cagaacatat gaggtttatg tgaattgctg 240ttatgttttt aagaagctta tcataagtaa tgaggttcat gatttttgac atagttagcc 300tccgcagtct ttcatttcaa gtaaataata gcgaaatatt ctttatactg aatacttata 360gtgaagcaaa gttctagctt tgagaaaatt ctttctgcaa ctaaatatag taaattacgg 420taaaatataa ataagtacat attgaagaaa atgagaca 458273050DNAArtificial SequenceStaphylococcus aureus 27aatttggtaa acctcaaaag gtaattacag atcaggcacc ttcaacgaag gtagcaatgg 60ctaaagtaat taaagctttt aaacttaaac ctgactgtca ttgtacatcg aaatatctga 120ataacctcat tgagcaagat caccgtcata ttaaagtaag aaagacaagg tatcaaagta 180tcaatacagc aaagaatact ttaaaaggta ttgaatgtat tcacgctcta tataaaaaga 240accgcaggtc tcttcagatc tacggatttt cgccatgcca cgaaattagc atcatgctag 300caagttaagc gaacactgac atgataaatt agtggttagc tatatttttt tactttgcaa 360cagaaccgaa aataatctct tcaatttatt tttatatgaa tcctgtgact caatgattgt 420aatatctaaa gatttcagtt catcatagac aatgttcttt tcaacatttt ttatagcaaa 480ttgattaaat aaattctcta atttctcccg tttgatttca ctaccataga ttatattatc 540attgatatag tcaatgaata atgacaaatt atcactcata acagtcccaa cccctttatt 600ttgatagact aattatcttc atcattgtaa aacaaattac accctttaaa tttaactcaa 660cttaaatatc gacaaattaa aaaacaataa aattacttga atattattca taatatatta 720acaactttat tatactgctc tttatatata aaatcattaa taattaaaca agccttaaaa 780tatttaactt ttttgtgatt attacacatt atcttatctg ctctttatca ccataaaaat 840agaaaaaaca agattcctaa agaatatagg aatcttgttt cagactgtgg acaaactgat 900tttttatcag ttagcttatt tagaaagttt tatttaaatt acagtttcta tttttattag 960atcacaattt tattttagct cttgttcaag taatcatttt tcgccaaaaa ctttatactg 1020aatagcttct acattaaata cttgtcaatg agatcatcta catctttaaa ttcagaataa 1080ttcgcatatg gatctataaa ataaaattgt ggttctttac cggaaacatt aaatattctt 1140aatattaaat atttctgctt atattctttc atagcaaaca tttcatttag cgacataaaa 1200aatggttcct caatactaga agatgtagat gttttaattt caataaattt ttctacagct 1260ttatctgtat ttgttggatc aaaagctact aaatcatagc catgaccgtg ttgagagcct 1320ggattatcat ttaaaatatt cctaaactgt tctttcttat cttcgtctat tttattatca 1380attagctcat taaagtaatt tagcgctaat ttttctccaa ctttaccggt taatttattc 1440tctttatttg atttttcaat ttctgaatca tttttagtag tctttgatac acctttttta 1500tattttggaa ttattccttt aggtgcttcc acttccttga gtgtcttatc tttttgtgct 1560gttctaattt cttcaatttc gctgtcttcc tgtatttcgt ctatgctatt gaccaagcta 1620tcataggatg tttttgtaac ttttgaagct aattcattaa atagttctaa aaatttcttt 1680aaatcctcta gcatatcttc ttctgtgaat ccttcattca aatcataata tttgaatctt 1740attgatccat gagaatatcc tgatggataa tcatttttta aatcataaga tgaatcttta 1800ttttctgcgt aataaaatct tccagtatta aattcatttg atgtaatata tttattgagt 1860tcggaagata aagttaatgc tctttgtttt gcagcatttt tatcccgcgg aaacatatca 1920cttatctttg accatccttg attcaaagat aagtatatgc cttctccttc cggatgaaaa 1980agatatacca aataatgtcc atcctttgtt tcttttgtta tattctcatc atatattgaa 2040atccaaggaa ctttactata gttcccagta gcaaccttcc ctacaactga atatttatct 2100tcttttatat gcacttttaa ctgcttgggt aacttatcat ggactaaagt tttatataga 2160tcacctttat cccaatcaga ttttttaact acattattgg tacgtttctc tttaattaat 2220ttaaggacct gcataaagtt gtctatcatt tgaaattccc tcctattata aaatatatta 2280tgtctcattt tcttcaatat gtacttattt atattttacc gtaatttact atatttagtt 2340gcagaaagaa ttttctcaaa gctagaactt tgcttcacta taagtattca gtataaagaa 2400tatttcgcta ttatttactt gaaatgaaag actgcggagg ctaactatgt caaaaatcat 2460gaacctcatt acttatgata agcttcttaa aaacataaca gcaattcaca taaacctcat 2520atgttctgat acattcaaaa tccctttatg aagcggctga aaaaaccgca tcatttatga 2580tatgcttctc ctcgcataat cttaaatgct ctgtacactt gttcaattaa cacaacccgc 2640atcatttgat gtgggaatgt cattttgctg aatgatagtg cgtagttact gcgttgtaag 2700acgtccttgt gcaggccgtt tgatccgcca atgacgaaaa caaagtcgct ttgcccttgg 2760gtcatgcgtt ggttcaattc ttgggccaat ccttcggaag atagcatctt tccttgtatt 2820tctaatgtaa tgactgtgga ttgtggtttg attttggcta gtattcgttg gccttctttt 2880tcttttactt gctcaatttc tttgtcactc atattttctg gtgctttttc gtctggaact 2940tctatgatgt ctatcttggt gtatgggcct aaacgttttt catattctgc tatggcttgc 3000ttccaatatt tctcttttag tttccctaca gctaaaatgg tgattttcat 3050281501DNAArtificial SequenceStaphylococcus aureus 28ttgcacaacc aattggtaaa gacaaagtga tggatccagc gaaacaacca gcgccaagta 60aagttgtatt gttgccagcg catagaggaa ctgttagtag tggtagagaa ggttctgatc 120gcgcattgga aggaactgct gtatcaagta agagcgggaa acaattggct agcatgtcag 180cgcctaaagg tagcacacat gagaagcagt taccaaaaac tggaactgat caaagttcaa 240gcccagcagc gatgtttgta ttagtagcag gtataggttt aattgcgact gtacgacgta 300gaaaagctag ctaaaatata ttgaaaacaa tactactgta tttcttaaac aagaggtacg 360gtagtgtttt tttatgaaaa aaagctataa ccgttgataa atatgggata taaaaacggg 420gataagtaat aagacatcaa ggtatttatc cacagaaatg gggatagtta tccagaattg 480tgtacaattt aaagagaaat acccacaatg cccacagagt tatccacaaa tacacaggtt 540atacactaaa aattgggcat gaatgtcaga aaaatatcaa aaactgcaaa gaatattggt 600ataataagag ggaacagtgt gaacaagtta ataacttgtg gataactgga aagttgataa 660caatttggag gaccaaacga catgaaaatc accattttag ctgtagggaa actaaaagag 720aaatattgga agcaagccat agcagaatat gaaaaacgtt taggcccata caccaagata 780gacatcatag aagttccaga cgaaaaagca ccagaaaata tgagcgacaa agaaattgag 840caagtaaaag aaaaagaagg ccaacgaata ctagccaaaa tcaaaccaca atcaacagtc 900attacattag aaatacaagg aaagatgcta tcttccgaag gattggccca agaattgaac 960caacgcatga cccaagggca aagcgacttt gtattcgtca ttggcggatc aaacggcctg 1020cacaaggacg tcttacaacg cagtaactac gcactatcat tcagcaaaat gacattccca 1080catcaaatga tgcgggttgt gttaattgaa caagtgtaca gagcatttaa gattatgcgt 1140ggagaagcat atcataaatg atgcggtttt ttcagccgct tcataaaggg attttgaatg 1200tatcagaaca tatgaggttt atgtgaattg ctgttatgtt tttaagaagc ttatcataag 1260taatgaggtt catgattttt gacatagtta gcctccgcag tctttcattt caagtaaata 1320atagcgaaat attctttata ctgaatactt atagtgaagc aaagttctag ctttgagaaa 1380attctttctg caactaaata tagtaaatta cggtaaaata taaataagta catattgaag 1440aaaatgagac ataatatatt ttataatagg agggaatttc aaatgataga caactttatg 1500c 150129917DNAArtificial SequenceStaphylococcus aureus 29aaatacaagg aaagatgcta tcttccgaag gattggccca agaattgaac caacgcatga 60cccaagggca aagcgacttt gtattcgtca ttggcggatc aaacggcctg cacaaggacg 120tcttacaacg tagtaactac gcactatcat tcagcaaaat gacattccca catcaaatga 180tgcgggttgt gttaattgag caagtgtata gagcatttaa gattatgcgt ggagaagcat 240atcataaatg atgcggtttt ttcagccgct tcataaaggg attttgaatg tatcagaaca 300tatgaggttt atgtgaattg ctgttatgtt tttaagaagc ttatcataag taatgaggtt 360catgattttt gacatagtta gcctccgcag tctttcattt caagtaaata atagcgaaat 420attctttata ctgaatactt atagtgaagc aaagttctag ctttgagaaa attctttctg 480caactaaata tagtaaatta cggtaaaata taaataagta catattgaag aaaatgagac 540ataatatatt ttataatagg agggaatttc aaatgataga caactttatg caggtcctta 600aattaattaa agagaaacgt accaataatg tagttaaaaa atctgattgg gataaaggtg 660atctatataa aactttagtc catgataagt tacccaagca gttaaaagtg catataaaag 720aagataaata ttcagttgta gggaaggttg ctactgggaa ctatagtaaa gttccttgga 780tttcaatata tgatgagaat ataacaaaag aaacaaagga tggatattat ttggtatatc 840tttttcatcc ggaaggagaa ggcatatact tatctttgaa tcaaggatgg tcaaagataa 900gtgatatgtt tccgcgg 917301132DNAArtificial SequenceStaphylococcus aureus 30gctgtaggga aactaaaaga gaaatattgg aagcaagcca tagcagaata tgaaaaacgt 60ttaggcccat acaccaagat agacatcata gaagttccag acgaaaaagc accagaaaat 120atgagcgaca aagaaattga gcaagtaaaa gaaaaagaag gccaacgaat actagccaaa 180atcaaaccac aatcaacagt cattacatta gaaatacaag gaaagatgct atcttccgaa 240ggattggccc aagaattgaa ccaacgcatg acccaagggc aaagcgactt tgtattcgtc 300attggcggat caaacggcct gcacaaggac gtcttacaac gtagtaacta cgcactatca 360ttcagcaaaa tgacattccc acatcaaatg atgcgggttg tgttaattga gcaagtgtat 420agagcattta agattatgcg tggagaagca tatcataaat gatgcggttt tttcagccgc 480ttcataaagg gattttgaat gtatcagaac atatgaggtt tatgtgaatt gctgttatgt 540ttttaagaag cttatcataa gtaatgaggt tcatgatttt tgacatagtt agcctccgca 600gtctttcatt tcaagtaaat aatagcgaaa tattctttat actgaatact tatagtgaag 660caaagttcta gctttgagaa aattctttct gcaactaaat atagtaaatt acggtaaaat 720ataaataagt acatattgaa gaaaatgaga cataatatat tttataatag gagggaattt 780caaatgatag acaactttat gcaggtcctt aaattaatta aagagaaacg taccaataat 840gtagttaaaa aatctgattg ggataaaggt gatctatata aaactttagt ccatgataag 900ttacccaagc agttaaaagt gcatataaaa gaagataaat attcagttgt agggaaggtt 960gctactggga actatagtaa agttccttgg atttcaatat atgatgagaa tataacaaaa 1020gaaacaaagg atggatatta tttggtatat ctttttcatc cggaaggaga aggcatatac 1080ttatctttga atcaaggatg gtcaaagata agtgatatgt ttccgcggga ta 1132311133DNAArtificial SequenceStaphylococcus aureus 31agctgtaggg aaactaaaag agaaatattg gaagcaagcc atagcagaat atgaaaaacg 60tttaggccca tacaccaaga tagacatcat agaagttcca gacgaaaaag caccagaaaa 120tatgagcgac aaagaaattg agcaagtaaa agaaaaagaa ggccaacgaa tactagccaa 180aatcaaacca caatcaacag tcattacatt agaaatacaa ggaaagatgc tatcttccga 240aggattggcc caagaattga accaacgcat gacccaaggg caaagcgact ttgtattcgt 300cattggcgga tcaaacggcc tgcacaagga cgtcttacaa cgtagtaact acgcactatc 360attcagcaaa atgacattcc cacatcaaat gatgcgggtt gtgttaattg agcaagtgta 420tagagcattt aagattatgc gtggagaagc atatcataaa tgatgcggtt ttttcagccg 480cttcataaag ggattttgaa tgtatcagaa catatgaggt ttatgtgaat tgctgttatg 540tttttaagaa gcttatcata agtaatgagg ttcatgattt ttgacatagt tagcctccgc 600agtctttcat ttcaagtaaa taatagcgaa atattcttta tactgaatac ttatagtgaa 660gcaaagttct agctttgaga aaattctttc tgcaactaaa tatagtaaat tacggtaaaa 720tataaataag tacatattga agaaaatgag acataatata ttttataata ggagggaatt 780tcaaatgata gacaacttta tgcaggtcct taaattaatt aaagagaaac gtaccaataa 840tgtagttaaa aaatctgatt gggataaagg tgatctatat aaaactttag tccatgataa 900gttacccaag cagttaaaag tgcatataaa agaagataaa tattcagttg tagggaaggt 960tgctactggg aactatagta aagttccttg gatttcaata tatgatgaga atataacaaa 1020agaaacaaag gatggatatt atttggtata tctttttcat ccggaaggag aaggcatata 1080cttatctttg aatcaaggat ggtcaaagat aagtgatatg tttccgcggg ata 1133321087DNAArtificial SequenceStaphylococcus aureus 32actaaaagag aaatattgga agcaagccat agcagaatat gaaaaacgtt taggcccata 60caccaagata gacatcatag aagttccaga cgaaaaagca ccagaaaata tgagcgacaa 120agaaattgag caagtaaaag aaaaagaagg ccaacgaata ctagccaaaa tcaaaccaca 180atcaacagtc attacattag aaatacaagg aaagatgcta tcttccgaag gattggcaca 240agaattgaac caacgcatga cccaagggca aagcgacttt gtattcgtca ttggcggatc 300aaacggcctg cacaaggacg tcttacaacg tagtaactac gcactatcat tcagcaaaat 360gacattccca catcaaatga tgcgggttgt gttaattgag caagtgtata gagcgtttaa 420gattatgcgt ggagaagcat atcataaatg atgcggtttt ttcagccgct tcataaaggg 480attttgaatg tatcagaaca tatgaggttt atgtgaattg ctgttatgtt tttaagaagc 540ttatcataag taatgaggtt catgattttt gacatagtta gcctccgcag tctttcattt 600caagtaaata atagcgaaat attctttata ctgaatactt atagtgaagc aaagttctag 660ctttgagaaa attctttctg caactaaata tagtaaatta cggtaaaata taaataagta 720catattgaag aaaatgagac ataatatatt ttataatagg agggaatttc aaatgataga 780caactttatg caggtcctta aattaattaa agagaaacgt accaataatg tagttaaaaa 840atctgattgg gataaaggtg atctatataa aactttagtc catgataagt tacccaagca 900gttaaaagtg catataaaag aagataaata ttcagttgta gggaaggttg ctactgggaa 960ctatagtaaa gttccttgga tttcaatata tgatgagaat ataacaaaag aaacaaagga 1020tggatattat ttggtatatc tttttcatcc ggaaggagaa ggcatatact tatctttgaa 1080tcaagga 108733903DNAArtificial SequenceStaphylococcus aureus 33caaggaaaga tgctatcttc cgaaggattg gcccaagaat tgaaccaacg catgacccaa 60gggcaaagcg actttgtatt cgtcattggc ggatcaaacg gcctgcacaa ggacgtctta 120caacgtagta actacgcact atcattcagc aaaatgacat tcccacatca aatgatgcgg 180gttgtgttaa ttgagcaagt gtatagagca tttaagatta tgcgtggaga agcatatcat 240aaatgatgcg gttttttcag ccgcttcata aagggatttt gaatgtatca gaacatatga 300ggtttatgtg aattgctgtt atgtttttaa gaagcttatc ataagtaatg aggttcatga 360tttttgacat agttagcctc cgcagtcttt catttcaagt aaataatagc gaaatattct 420ttatactgaa tacttatagt gaagcaaagt tctagctttg agaaaattct ttctgcaact 480aaatatagta aattacggta aaatataaat aagtacatat tgaagaaaat gagacataat 540atattttata ataggaggga atttcaaatg atagacaact ttatgcaggt ccttaaatta 600attaaagaga aacgtaccaa taatgtagtt aaaaaatctg attgggataa aggtgatcta 660tataaaactt tagtccatga taagttaccc aagcagttaa aagtgcatat aaaagaagat 720aaatattcag ttgtagggaa ggttgctact gggaactata gtaaagttcc ttggatttca 780atatatgatg agaatataac aaaagaaaca aaggatggat attatttggt atatcttttt 840catccggaag gagaaggcat atacttatct ttgaatcaag gatggtcaaa gataagtgat 900atg 903341114DNAArtificial SequenceStaphylococcus aureus 34ggaaactaaa agagaaatat tggaagcaag ccatagcaga atatgaaaaa cgtttaggcc 60catacaccaa gatagacatc atagaagttc cagacgaaaa agcaccagaa aatatgagcg 120acaaagaaat tgagcaagta aaagaaaaag aaggccaacg aatactagcc aaaatcaaac 180cacaatcaac agtcattaca ttagaaatac aaggaaagat gctatcttcc gaaggattgg 240cccaagaatt gaaccaacgc atgacccaag ggcaaagcga ctttgtattc gtcattggcg 300gatcaaacgg cctgcacaag gacgtcttac aacgtagtaa ctacgcacta tcattcagca 360aaatgacatt cccacatcaa atgatgcggg ttgtgttaat tgagcaagtg tatagagcat 420ttaagattat gcgtggagaa gcatatcata aatgatgcgg ttttttcagc cgcttcataa 480agggattttg aatgtatcag aacatatgag gtttatgtga attgctgtta tgtttttaag 540aagcttatca taagtaatga ggttcatgat ttttgacata gttagcctcc gcagtctttc 600atttcaagta aataatagcg aaatattctt tatactgaat acttatagtg aagcaaagtt 660ctagctttga gaaaattctt tctgcaacta aatatagtaa attacggtaa aatataaata 720agtacatatt gaagaaaatg agacataata tattttataa taggagggaa tttcaaatga 780tagacaactt tatgcaggtc cttaaattaa ttaaagagaa acgtaccaat aatgtagtta 840aaaaatctga ttgggataaa ggtgatctat ataaaacttt agtccatgat aagttaccca 900agcagttaaa agtgcatata aaagaagata aatattcagt tgtagggaag gttgctactg 960ggaactatag taaagttcct tggatttcaa tatatgatga gaatataaca aaagaaacaa 1020aggatggata ttatttggta tatctttttc atccggaagg agaaggcata tacttatctt 1080tgaatcaagg atggtcaaag ataagtgata tgtt 1114351121DNAArtificial SequenceStaphylococcus aureus 35ggaaactaaa agagaaatat tggaagcaag ccatagcaga atatgaaaaa cgtttaggcc 60catacaccaa gatagacatc atagaagttc cagacgaaaa agcaccagaa aatatgagcg 120acaaagaaat tgagcaagta aaagaaaaag aaggccaacg aatactagcc aaaatcaaac 180cacaatccac agtcattaca ttagaaatac aaggaaagat gctatcttcc gaaggattgg 240cccaagaatt gaaccaacgc atgacccaag ggcaaagcga ctttgtattc gtcattggcg 300gatcaaacgg cctgcacaag gacgtcttac aacgcagtaa ctatgcacta tcatttagca 360aaatgacatt cccacatcaa atgatgcggg ttgtgttaat tgaacaagtg tatagagcat 420ttaagattat gcgtggagaa gcatatcata aatgatgcgg ttttttcagc cgcttcataa 480agggattttg aatgtatcag aacatatgag gtttatgtga attgctgtta tgtttttaag 540aagcttatca taagtaatga ggttcatgat ttttgacata gttagcctcc gcagtctttc 600atttcaagta aataatagcg aaatattctt tatactgaat acttatagtg aagcaaagtt 660ctagctttga gaaaattctt tctgcaacta aatatagtaa attacggtaa aatataaata 720agtacatatt gaagaaaatg agacataata tattttataa taggagggaa tttcaaatga 780tagacaactt tatgcaggtc cttaaattaa ttaaagagaa acgtaccaat aatgtagtta 840aaaaatctga ttgggataaa ggtgatctat ataaaacttt agtccatgat aagttaccca 900agcagttaaa agtgcatata aaagaagata aatattcagt tgtagggaag gttgctactg 960ggaactatag taaagttcct tggatttcaa tatatgatga gaatataaca aaagaaacaa 1020aggatggata ttatttggta tatctttttc atccggaagg agaaggcata tacttatctt 1080tgaatcaagg atggtcaaag ataagtgata tgtttccgcg g 1121361121DNAArtificial SequenceStaphylococcus aureus 36tagctgtagg gaaactaaaa gagaaatatt ggaagcaagc catagcagaa tatgaaaaac 60gtttaggccc atacaccaag atagacatca tagaagttcc agacgaaaaa gcaccagaaa 120atatgagcga caaagaaatt gagcaagtaa aagaaaaaga aggccaacga atactagcca 180aaatcaaacc acaatccaca gtcattacat tagaaataca aggaaagatg ctatcttccg 240aaggattggc ccaagaattg aaccaacgca tgacccaagg gcaaagcgac tttgtattcg 300tcattggcgg atcaaacggc ctgcacaagg acgtcttaca acgcagtaac tatgcactat 360catttagcaa aatgacattc ccacatcaaa tgatgcgggt tgtgttaatt gaacaagtgt 420atagagcatt taagattatg cgtggagaag catatcataa atgatgcggt tttttcagcc 480gcttcataaa gggattttga atgtatcaga acatatgagg tttatgtgaa ttgctgttat 540gtttttaaga agcttatcat aagtaatgag gttcatgatt tttgacatag ttagcctccg 600cagtctttca tttcaagtaa ataatagcga aatattcttt atactgaata cttatagtga 660agcaaagttc tagctttgag aaaattcttt ctgcaactaa atatagtaaa ttacggtaaa 720atataaataa gtacatattg aagaaaatga gacataatat attttataat aggagggaat 780ttcaaatgat agacaacttt atgcaggtcc ttaaattaat taaagagaaa cgtaccaata 840atgtagttaa aaaatctgat tgggataaag gtgatctata taaaacttta gtccatgata 900agttacccaa gcagttaaaa gtgcatataa aagaagataa atattcagtt gtagggaagg 960ttgctactgg gaactatagt aaagttcctt ggatttcaat atatgatgag aatataacaa 1020aagaaacaaa ggatggatat tatttggtat atctttttca tccggaagga gaaggcatat 1080acttatcttt gaatcaagga tggtcaaaga taagtgatat g 1121371131DNAArtificial SequenceStaphylococcus aureus 37ctgtagggaa actaaaagag aaatattgga agcaagccat agcagaatat gaaaaacgtt 60taggcccata caccaagata gacatcatag aagttccaga cgaaaaagca ccagaaaata 120tgagcgacaa agaaattgag caagtaaaag aaaaagaagg ccaacgaata ctagccaaaa 180tcaaaccaca

atccacagtc attacattag aaatacaagg aaagatgcta tcttccgaag 240gattggccca agaattgaac caacgcatga cccaagggca aagcgacttt gtattcgtca 300ttggcggatc aaacggcctg cacaaggacg tcttacaacg cagtaactat gcactatcat 360ttagcaaaat gacattccca catcaaatga tgcgggttgt gttaattgaa caagtgtata 420gagcatttaa gattatgcgt ggagaagcat atcataaatg atgcggtttt ttcagccgct 480tcataaaggg attttgaatg tatcagaaca tatgaggttt atgtgaattg ctgttatgtt 540tttaagaagc ttatcataag taatgaggtt catgattttt gacatagtta gcctccgcag 600tctttcattt caagtaaata atagcgaaat attctttata ctgaatactt atagtgaagc 660aaagttctag ctttgagaaa attctttctg caactaaata tagtaaatta cggtaaaata 720taaataagta catattgaag aaaatgagac ataatatatt ttataatagg agggaatttc 780aaatgataga caactttatg caggtcctta aattaattaa agagaaacgt accaataatg 840tagttaaaaa atctgattgg gataaaggtg atctatataa aactttagtc catgataagt 900tacccaagca gttaaaagtg catataaaag aagataaata ttcagttgta gggaaggttg 960ctactgggaa ctatagtaaa gttccttgga tttcaatata tgatgagaat ataacaaaag 1020aaacaaagga tggatattat ttggtatatc tttttcatcc ggaaggagaa ggcatatact 1080tatctttgaa tcaaggatgg tcaaagataa gtgatatgtt tccgcgggat a 113138896DNAArtificial SequenceStaphylococcus aureus 38cattagaaat acaaggaaag atgctatctt ccgaaggatt ggcccaagaa ttgaaccaac 60gcatgaccca agggcaaagc gactttgtat tcgtcattgg cggatcaaac ggcctgcaca 120aggacgtctt acaacgcagt aactatgcac tatcatttag caaaatgaca ttcccacatc 180aaatgatgcg ggttgtgtta attgaacaag tgtatagagc atttaagatt atgcgtggag 240aagcatatca taaatgatgc ggttttttca gccgcttcat aaagggattt tgaatgtatc 300agaacatatg aggtttatgt gaattgctgt tatgttttta agaagcttat cataagtaat 360gaggttcatg atttttgaca tagttagcct ccgcagtctt tcatttcaag taaataatag 420cgaaatattc tttatactga atacttatag tgaagcaaag ttctagcttt gagaaaattc 480tttctgcaac taaatatagt aaattacggt aaaatataaa taagtacata ttgaagaaaa 540tgagacataa tatattttat aataggaggg aatttcaaat gatagacaac tttatgcagg 600tccttaaatt aattaaagag aaacgtacca ataatgtagt taaaaaatct gattgggata 660aaggtgatct atataaaact ttagtccatg ataagttacc caagcagtta aaagtgcata 720taaaagaaga taaatattca gttgtaggga aggttgctac tgggaactat agtaaagttc 780cttggatttc aatatatgat gagaatataa caaaagaaac aaaggatgga tattatttgg 840tatatctttt tcatccggaa ggagaaggca tatacttatc tttgaatcaa ggatgg 896391125DNAArtificial SequenceStaphylococcus aureus 39ggaaactaaa agagaaatat tggaagcaag ccatatcaga atatgaaaaa cgtttaggcc 60catacaccaa gatagacatc atagaagttc cagacgaaaa agcaccagaa aatatgagcg 120acaaagaaat cgagcaagta aaagaaaaag aaggccaacg aatactagcc aaaatcaaac 180cacaatcaac agtcattaca ttagaaatac aaggaaagat gctatcttcc gaaggattgg 240ctcaagaatt gaaccaacgc atgacccaag ggcaaagcga ctttgtattc gttattggcg 300gatcaaacgg cctgcacaag gacgtcttac aacgcagtaa ctatgcacta tcattcagca 360aaatgacatt tccacatcag atgatgcggg ttgtgttaat tgagcaagtg tatagagcat 420ttaagattat gcgtggggaa gcatatcata aatgatgcgg ttttttcagc cgcttcataa 480agggattttg aatgtatcag aacatatgag gtttatgtga attgctgtta tgtttttaag 540aagcttatca taagtaatga ggttcatgat ttttgacata gttagcctcc gcagtctttc 600atttcaagta aataatagcg aaatattctt tatactgaat acttatagtg aagcaaagtt 660ctagctttga gaaaattctt tctgcaacta aatatagtaa attacggtaa aatataaata 720agtacatatt gaagaaaatg agacataata tattttataa taggagggaa tttcaaatga 780tagacaactt tatgcaggtc cttaaattaa ttaaagagaa acgtaccaat aatgtagtta 840aaaaatctga ttgggataaa ggtgatctat ataaaacttt agtccatgat aagttaccca 900agcagttaaa agtgcatata aaagaagata aatattcagt tgtagggaag gttgctactg 960ggaactatag taaagttcct tggatttcaa tatatgatga gaatataaca aaagaaacaa 1020aggatggata ttatttggta tatctttttc atccggaagg agaaggcata tacttatctt 1080tgaatcaagg atggtcaaag ataagtgata tgtttccgcg ggata 1125401125DNAArtificial SequenceStaphylococcus aureus 40ggaaactaaa agagaaatat tggaagcaag ccatagcaga atatgaaaaa cgtttaggcc 60catacaccaa gatagacatc atagaagttc cagacgaaaa agcaccagaa aatatgagcg 120acaaagaaat tgagcaagta aaagaaaaag aaggccaacg aatactagcc aaaatcaaac 180cacaatcaac agtcattaca ttagaaatac aaggaaagat gctatcttcc gaaggattgg 240cacaagaatt gaaccaacgc atgacccaag ggcaaagcga ctttgtattc gtcattggcg 300gatcaaacgg cctgcacaag gacgtcttac aacgtagtaa ctacgcacta tcattcagca 360aaatgacatt cccacatcaa atgatgcggg ttgtgttaat tgagcaagtg tatagagcgt 420ttaagattat gcgtggagaa gcatatcata aatgatgcgg ttttttcagc cgcttcataa 480agggattttg aatgtatcag aacatatgag gtttatgtga attgctgtta tgtttttaag 540aagcttatca taagtaatga ggttcatgat ttttgacata gttagcctcc gcagtctttc 600atttcaagta aataatagcg aaatattctt tatactgaat acttatagtg aagcaaagtt 660ctagctttga gaaaattctt tctgcaacta aatatagtaa attacggtaa aatataaata 720agtacatatt gaagaaaatg agacataata tattttataa taggagggaa tttcaaatga 780tagacaactt tatgcaggtc cttaaattaa ttaaagagaa acgtaccaat aatgtagtta 840aaaaatctga ttgggataaa ggtgatctat ataaaacttt agtccatgat aagttaccca 900agcagttaaa agtgcatata aaagaagata aatattcagt tgtagggaag gttgctactg 960ggaactatag taaagttcct tggatttcaa tatatgatga gaatataaca aaagaaacaa 1020aggatggata ttatttggta tatctttttc atccggaagg agaaggcata tacttatctt 1080tgaatcaagg atggtcaaag ataagtgata tgtttccgcg ggata 112541926DNAArtificial SequenceStaphylococcus aureus 41tacattagaa atacaaggaa agatgctatc ttccgaagga ttggcccaag aattgaacca 60acgcatgacc caagggcaaa gcgactttgt attcgtcatt ggcggatcaa acggcctgca 120caaggacgtc ttacaacgca gtaactatgc actatcattt agcaaaatga cattcccaca 180tcaaatgatg cgggttgtgt taattgaaca agtgtataga gcatttaaga ttatgcgtgg 240agaagcatat cataaatgat gcggtttttt cagccgcttc ataaagggat tttgaatgta 300tcagaacata tgaggtttat gtgaattgct gttatgtttt taagaagctt atcataagta 360atgaggttca tgatttttga catagttagc ctccgcagtc tttcatttca agtaaataat 420agcgaaatat tctttatact gaatacttat agtgaagcaa agttctagct ttgagaaaat 480tctttctgca actaaatata gtaaattacg gtaaaatata aataagtaca tattgaagaa 540aatgagacat aatatatttt ataataggag ggaatttcaa atgatagaca actttatgca 600ggtccttaaa ttaattaaag agaaacgtac caataatgta gttaaaaaat ctgattggga 660taaaggtgat ctatataaaa ctttagtcca tgataagtta cccaagcagt taaaagtgca 720tataaaagaa gataaatatt cagttgtagg gaaggttgct actgggaact atagtaaagt 780tccttggatt tcaatatatg atgagaatat aacaaaagaa acaaaggatg gatattattt 840ggtatatctt tttcatccgg aaggagaagg catatactta tctttgaatc aaggatggtc 900aaagataagt gatatgtttc cgcggg 92642928DNAArtificial SequenceStaphylococcus aureus 42tacattagaa atacaaggaa agatgctatc ttccgaagga ttggcccaag aattgaacca 60acgcatgacc caagggcaaa gcgactttgt attcgtcatt ggcggatcaa acggcctgca 120caaggacgtc ttacaacgca gtaactacgc actatcattc agcaaaatga cattcccaca 180tcaaatgatg cgggttgtgt taattgaaca agtgtacaga gcatttaaga ttatgcgtgg 240agaagcatat cataaatgat gcggtttttt cagccgcttc ataaagggat tttgaatgta 300tcagaacata tgaggtttat gtgaattgct gttatgtttt taagaagctt atcataagta 360atgaggttca tgatttttga catagttagc ctccgcagtc tttcatttca agtaaataat 420agcgaaatat tctttatact gaatacttat agtgaagcaa agttctagct ttgagaaaat 480tctttctgca actaaatata gtaaattacg gtaaaatata aataagtaca tattgaagaa 540aatgagacat aatatatttt ataataggag ggaatttcaa atgatagaca actttatgca 600ggtccttaaa ttaattaaag agaaacgtac caataatgta gttaaaaaat ctgattggga 660taaaggtgat ctatataaaa ctttagtcca tgataagtta cccaagcagt taaaagtgca 720tataaaagaa gataaatatt cagttgtagg gaaggttgct actgggaact atagtaaagt 780tccttggatt tcaatatatg atgagaatat aacaaaagaa acaaaggatg gatattattt 840ggtatatctt tttcatccgg aaggagaagg catatactta tctttgaatc aaggatggtc 900aaagataagt gatatgtttc cgcgggat 92843479DNAArtificial SequenceStaphylococcus aureus 43ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaaca 47944480DNAArtificial SequenceStaphylococcus aureus 44ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataac atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaacag 48045480DNAArtificial SequenceStaphylococcus aureus 45ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac atccccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agatttgtgt tagaaacagt 48046480DNAArtificial SequenceStaphylococcus aureus 46ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaacag 48047309DNAUnknownStaphylococcus aureus 47ggcggatcaa acggcctgca caaggacgtc ttacaacgca gtaactacgc actatcattc 60agcaaaatga cattcccaca tcaaatgatg cgggttgtgt taattgaaca agtgtacaga 120gcatttaaga ttatgcgtgg agaagcgtat cataaataaa actaaaaatt aggttgtgta 180taatttaaaa atctaatgag atgtggagga attacatata tgaaatattg gattatncct 240tgcaatatca tacgatgttt atagagtgtt taataaacca tttttcaact attgatgatc 300tacaatata 30948471DNAArtificial SequenceStaphylococcus aureus 48ttggcggatc aaacggcctg cacaaggacg tcttacaacg cagtaactac gcactatcat 60tcagcaaaat gacattccca catcaaatga tgcgggttgt gttaattgaa caagtgtaca 120gagcatttaa gattatgcgt ggagaagcgt atcataaata aaactaaaaa ttaggttgtg 180tataatttaa aaatttaatg agatgtggag gaattacata tatgaaatat tggattatac 240cttgcaatat catacgatgt ttatagagtg tttaataaac catttttcaa ctattgatga 300tctagaatat ataataactg tacaaattat attgattatg gaactacaat taaattaaga 360aattgatgat gaaattttaa atttaaacta atggaatcaa gaaagaatga aaggaaatat 420acaatgccta cgattaataa aaggaagttt attagatttt gtgttagaaa c 47149480DNAArtificial SequenceStaphylococcus aureus 49ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaacag 48050480DNAArtificial SequenceStaphylococcus aureus 50ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaacag 48051480DNAArtificial SequenceStaphylococcus aureus 51ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaacag 48052478DNAArtificial SequenceStaphylococcus aureus 52ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaac 47853479DNAArtificial SequenceStaphylococcus aureus 53ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaaca 47954480DNAArtificial SequenceStaphylococcus aureus 54ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcncgaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaacag 48055480DNAArtificial SequenceStaphylococcus aureus 55ttcgtcattg gcggatcaaa cggcctgcac aaggacgtct tacaacgcag taactacgca 60ctatcattca gcaaaatgac attcccacat caaatgatgc gggttgtgtt aattgaacaa 120gtgtacagag catttaagat tatgcgtgga gaagcgtatc ataaataaaa ctaaaaatta 180ggttgtgtat aatttaaaaa tttaatgaga tgtggaggaa ttacatatat gaaatattgg 240attatacctt gcaatatcat acgatgttta tagagtgttt aataaaccat ttttcaacta 300ttgatgatct agaatatata ataactgtac aaattatatt gattatggaa ctacaattaa 360attaagaaat tgatgatgaa attttaaatt taaactaatg gaatcaagaa agaatgaaag 420gaaatataca atgcctacga ttaataaaag gaagtttatt agattttgtg ttagaaacag 480561256DNAArtificial SequenceStaphylococcus aureus 56ttcagaaaaa tgattaatgt gtttcaataa aatctctcct tctttgtgaa catattcatt 60tttatactaa ttaatataat ttccaaaaaa gtttctgttt aaaagtgaaa aatattattt 120accgtttgac ttaaatcttc aatatatagg tgtttatatg tatcattttg cgccaatttg 180aataaacggg aatcaagtct gtttctgagt ttatttcaac tttcttatag taaacattgt 240cttaatatga tgaacttcaa taaaactttc cctatgcccc ataaaatttt ctcaaaatca 300aaaataacat accttacaac ttttaccgtc gatatcaatt gctcttttct taatttagga 360ttgctttcaa attttgtact ataacgtgaa actacttttc cttctttata attaaaattt 420actaattcac aatcattttt acttccattt acaaaaacat ccactgtttc taacacaaaa 480tctaataaac ttccttttat taatcgtagg cattgtatat ttcctttcat tctttcttga 540ttccattagt ttaaatttaa aatttcatcc atcaatttct taatttaatt gtagttccat 600aatcaatata atttgtacag ttattatata ttctagatca tcaatagttg aaaaatggtt 660tattaaacac tctataaaca tcgtatgata ttgcaaggta taatccaata tttcatatat 720gtaattcctc cacatctcat taaattttta aattatacac aacctaattt ttagttttat 780ttatgatacg cttctccacg cataatctta aatgctctgt acacttgttc aattaacaca 840acccgcatca tttgatgtgg gaatgtcatt ttgctgaatg atagtgcgta gttactgcgt 900tgtaagacgt ccttgtgcag gccgtttgat ccgccaatga cgaatacaaa gtcgctttgc 960ccttgggtca tgcgttggtt caattcttgg gccaatcctt cggaagatag catctttcct 1020tgtatttcta atgtaatgac tgttgattgt ggtttgattt tggctagtat tcgttggcct 1080tctttttctt ttacttgctc aatttctttg tcgctcatat tttctggtgc tttttcgtct 1140ggaacttcta tgatgtctat cttggtgtat gggcctaaac gtttttcata ttctgctatg 1200gcttgcttcc aatatttctc ttttagtttc cctacagcta aaatggtgat tttcat 125657679DNAArtificial SequenceStaphylococcus aureus 57ataagaggga acagtgtgaa caagttaata acttgtggat aactggaaag ttgataacaa 60tttggaggac caaacgacat gaaaatcacc attttagctg tagggaaact aaaagagaaa 120tattggaagc aagccatagc agaatatgaa aaacgtttag gcccatacac caagatagac 180atcatagaag ttccagacga aaaagcacca gaaaatatga gcgacaaaga aattgagcaa 240gtaaaagaaa aagaaggcca acgaatacta gccaaaatca aaccacaatc cacagtcatt 300acattagaaa tacaaggaaa gatgctatct tccgaaggat tggcccaaga attgaaccaa 360cgcatgaccc aagggcaaag cgactttgta ttcgtcattg gcggatcaaa cggcctgcac 420aaggacgtct tacaacgcag taactatgca

ctatcattta gcaaaatgac attcccacat 480caaatgatgc gggttgtgtt aattgaacaa gtgtatagag catttaagat tatgcgtgga 540gaggcttatc ataaataaaa ctaaaaatta gattgtgtat aatttaaaaa tttaatgaga 600tgtggaggaa ttacatatat gaaatattgg agtatacctt gcaatatcat acgatgttta 660tagagtgttt aataaacca 67958782DNAArtificial SequenceStaphylococcus aureus 58caatgcccac agagttatcc acaaatacac aggttataca ctaaaaattg ggcatgaatg 60tcagaaaaat atcaaaaact gcaaagaata ttggtataat aagagggaac agtgtgaaca 120agttaataac ttgtggataa ctggaaagtt gataacaatt tggaggacca aacgacatga 180aaatcaccat tttagctgta gggaaactaa aagagaaata ttggaagcaa gccatagcag 240aatatgaaaa acgtttaggc ccatacacca agatagacat catagaagtt ccagacgaaa 300aagcaccaga aaatatgagc gacaaagaaa ttgagcaagt aaaagaaaaa gaaggccaac 360gaatactagc caaaatcaaa ccacaatcaa cagtcattac attagaaata caaggaaaga 420tgctatcttc cgaaggattg gcccaagaat tgaaccaacg catgacccaa gggcaaagcg 480actttgtatt cgtcattggc ggatcaaacg gcctgcacaa ggacgtctta caacgcagta 540actacgcact atcattcagc aaaatgacat tcccacatca aatgatgcgg gttgtgttaa 600ttgaacaagt gtacagagca tttaagatta tgcgtggaga agcgtatcat aaataaaact 660aaaaattagg ttgtgtataa tttaaaaatt taatgagatg tggaggaatt acatatatga 720aatattggat tataccttgc aatatcatac gatgtttata gagtgtttaa taaaccattt 780tt 782591045DNAArtificial SequenceStaphylococcus aureus 59ccagtttttt gtttaatgaa caaggtaaat tacgagataa tatttgaaga aaacaataaa 60gtagagatgg atttccatat cctctttagt agcggttttt atctgtaagg tttattaata 120attaaataaa taggcgggat agttatatat agcttattaa tgaaagaata tgattattaa 180tttagtatta tattttaata ttaaaaagaa gatatgaaat aattattcat accttccacc 240ttacaataat tagttttcaa tcgaatatta agattattag tagtcttaaa agttaagact 300tccttatatt aatgacctaa tttattattt gcctcatgaa ttatcttttt atttctttga 360tatgtcccaa accacatcgt gatatacact acaataaata ttatgatgaa actaataata 420ttctcaaagt tcagatggaa ccaacctgct agaatagcga gtgggaagaa taggattatc 480atcaatataa agtgaactac agtctgtttt gttatactcc aatcggtatc tgtaaatatc 540aaattaccat aagtaaacaa aattccaatc aatgcccata gtgctacaca tattagcata 600ataaccgctt cattaaagtt ttcataataa attttaccca taaaagaatc tggatatagt 660ggtacatatt tatcccttga aaaaaataag tgaagtaatg acagaaatca taagaccagt 720gaacgcacct ttttgaacag cgtggaataa ttttttcata gtgagatgga ccattccatt 780tgtttctaac ttcaagtgat caatgtaatt tagattgata atttctgatt ttgaaatacg 840cacgaatatt gaaccgacaa gctcttcaat ttggtaaagt cgctgataaa gttttaaagc 900tttattattc attgttatcg catacctgtt tatcttctac tatgaactgt gcaatttgtt 960ctagatcaat tgggtaaaca tgatggttct gttgcaaagt aaaaaaatat agctaaccac 1020taatttatca tgtcagtgtt cgctt 1045601118DNAArtificial SequenceStaphylococcus aureus 60cagagcattt aagattatgc gtggagaagc gtaccacaaa tgatgcggtt ttttatccag 60ttttttgttt aatgaacaag gtaaattacg agataatatt tgaagaaaac aataaagtag 120agatggattt ccatatcctc tttagtagcg gtttttatct gtaaggttta ttaataatta 180aataaatagg cgggatagtt atatatagct tattaatgaa agaatatgat tattaattta 240gtattatatt ttaatattaa aaagaagata tgaaataatt attcatacct tccaccttac 300aataattagt tttcaatcga atattaagat tattagtagt cttaaaagtt aagacttcct 360tatattaatg acctaattta ttatttgcct catgaattat ctttttattt ctttgatatg 420tcccaaacca catcgtgata tacactacaa taaatattat gatgaaacta ataatattct 480caaagttcag atggaaccaa cctgctagaa tagcgagtgg gaagaatagg attatcatca 540atataaagtg aactacagtc tgttttgtta tactccaatc ggtatctgta aatatcaaat 600taccataagt aaacaaaatt ccaatcaatg cccatagtgc tacacatatt agcataataa 660ccgcttcatt aaagttttca taataaattt tacccataaa agaatctgga tatagtagta 720catatttatc ccttgaaaaa aataagtgaa gtaatgacag aaatcataag accagtgaac 780gcaccttttt gaacagcgtg gaataatttt ttcatagtga gatggaccat tccatttgtt 840tctaacttca agtgatcaat gtaatttaga ttgataattt ctgattttga aatacgcacg 900aatattgaac cgacaagctc ttcaatttgg taaagtcgct gataaagttt taaagcttta 960ttattcattg ttatcgcata cctgtttatc ttctactatg aactgtgcaa tttgttctag 1020atcaattggg taaacatgat ggttctgttg caaagtaaaa aaatatagct aaccactaat 1080ttatcatgtc agtgttcgct taacttgcta gcatgatg 1118611118DNAArtificial SequenceStaphylococcus aureus 61cagagcattt aagattatgc gtggagaagc gtaccacaaa tgatgcggtt ttttatccag 60ttttttgttt aatgaacaag gtaaattacg agataatatt tgaagaaaac aataaagtag 120agatggattt ccatatcctc tttagtagcg gtttttatct gtaaggttta ttaataatta 180aataaatagg cgggatagtt atatatagct tattaatgaa agaatatgat tattaattta 240gtattatatt ttaatattaa aaagaagata tgaaataatt attcatacct tccaccttac 300aataattagt tttcaatcga atattaagat tattagtagt cttaaaagtt aagacttcct 360tatattaatg acctaattta ttatttgcct catgaattat ctttttattt ctttgatatg 420tcccaaacca catcgtgata tacactacaa taaatattat gatgaaacta ataatattct 480caaagttcag atggaaccaa cctgctagaa tagcgagtgg gaagaatagg attatcatca 540atataaagtg aactacagtc tgttttgtta tactccaatc ggtatctgta aatatcaaat 600taccataagt aaacaaaatt ccaatcaatg cccatagtgc tacacatatt agcataataa 660ccgcttcatt aaagttttca taataaattt tacccataaa agaatctgga tatagtagta 720catatttatc ccttgaaaaa aataagtgaa gtaatgacag aaatcataag accagtgaac 780gcaccttttt gaacagcgtg gaataatttt ttcatagtga gatggaccat tccatttgtt 840tctaacttca agtgatcaat gtaatttaga ttgataattt ctgattttga aatacgcacg 900aatattgaac cgacaagctc ttcaatttgg taaagtcgct gataaagttt taaagcttta 960ttattcattg ttatcgcata cctgtttatc ttctactatg aactgtgcaa tttgttctag 1020atcaattggg taaacatgat ggttctgttg caaagtaaaa aaatatagct aaccactaat 1080ttatcatgtc agtgttcgct taacttgcta gcatgatg 1118621113DNAArtificial SequenceStaphylococcus aureus 62agcatttaag attatgcgtg gagaagcgta ccacaaatga tgcggttttt tatccagttt 60tttgtttaat gaacaaggta aattacgaga taatatttga agaaaacaat aaagtagaga 120tggatttcca tatcctcttt agtagcggtt tttatctgta aggtttatta ataattaaat 180aaataggcgg gatagttata tatagcttat taatgaaaga atatgattat taatttagta 240ttatatttta atattaaaaa gaagatatga aataattatt cataccttcc accttacaat 300aattagtttt caatcgaata ttaagattat tagtagtctt aaaagttaag acttccttat 360attaatgacc taatttatta tttgcctcat gaattatctt tttatttctt tgatatgtcc 420caaaccacat cgtgatatac actacaataa atattatgat gaaactaata atattctcaa 480agttcagatg gaaccaacct gctagaatag cgagtgggaa gaataggatt atcatcaata 540taaagtgaac tacagtctgt tttgttatac tccaatcggt atctgtaaat atcaaattac 600cataagtaaa caaaattcca atcaatgccc atagtgctac acatattagc ataataaccg 660cttcattaaa gttttcataa taaattttac ccataaaaga atctggatat agtggtacat 720atttatccct tgaaaaaaat aagtgaagta atgacagaaa tcataagacc agtgaacgca 780cctttttgaa cagcgtggaa taattttttc atagtgagat ggaccattcc atttgtttct 840aacttcaagt gatcaatgta atttagattg ataatttctg attttgaaat acgcacgaat 900attgaaccga caagctcttc aatttggtaa agtcgctgat aaagttttaa agctttatta 960ttcattgtta tcgcatacct gtttatcttc tactatgaac tgtgcaattt gttctagatc 1020aattgggtaa acatgatggt tctgttgcaa agtaaaaaaa tatagctaac cactaattta 1080tcatgtcagt gttcgcttaa cttgctagca tga 1113632153DNAArtificial SequenceStaphylococcus aureus 63ctgtagggaa actaaaagag aaatactgga agcaagccat agcagaatat gaaaaacgtt 60taggcccata caccaagata gacatcatag aagttccaga cgaaaaagca ccagaaaata 120tgagcgacaa agaaatcgag caagtaaaag aaaaagaagg ccaacgaata ctagccaaaa 180tcaaaccaca atccacagtc attacattag aaatacaagg aaagatgcta tcttccgaag 240gattggccca agaattgaac caacgcatga cccaagggca aagcgacttt gtattcgtca 300ttggcggatc aaacggcctg cacaaggacg tcttacaacg cagtaactac gcactatcat 360tcagcaaaat gacattccca catcaaatga tgcgggttgt gttaattgaa caagtgtaca 420gagcatttaa gattatgcgt ggagaagcgt accacaaatg atgcggtttt ttatccagtt 480ttttgtttaa tgaacaaggt aaattacgag ataatatttg aagaaaacaa taaagtagag 540atggatttcc atatcctctt tagtagcggt ttttatctgt aaggtttatt aataattaaa 600taaataggcg ggatagttat atatagctta ttaatgaaag aatatgatta ttaatttagt 660attatatttt aatattaaaa agaagatatg aaataattat tcataccttc caccttacaa 720taattagttt tcaatcgaat attaagatta ttagtagtct taaaagttaa gacttcctta 780tattaatgac ctaatttatt atttgcctca tgaattatct ttttatttct ttgatatgtc 840ccaaaccaca tcgtgatata cactacaata aatattatga tgaaactaat aatattctca 900aagttcagat ggaaccaacc tgctagaata gcgagtggga agaataggat tatcatcaat 960ataaagtgaa ctacagtctg ttttgttata ctccaatcgg tatctgtaaa tatcaaatta 1020ccataagtaa acaaaattcc aatcaatgcc catagtgcta cacatattag cataataacc 1080gcttcattaa agttttcata ataaatttta cccataaaag aatctggata tagtggtaca 1140tatttatccc ttgaaaaaaa taagtgaagt aatgacagaa atcataagac cagtgaacgc 1200acctttttga acagcgtgga ataatttttt catagtgaga tggaccattc catttgtttc 1260taacttcaag tgatcaatgt aatttagatt gataatttct gattttgaaa tacgcacgaa 1320tattgaaccg acaagctctt caatttggta aagtcgctga taaagtttta aagctttatt 1380attcattgtt atcgcatacc tgtttatctt ctactatgaa ctgtgcaatt tgttctagat 1440caattgggta aacatgatgg ttctgttgca aagtaaaaaa atatagctaa ccactaattt 1500atcatgtcag tgttcgctta acttgctagc atgatgctaa tttcgtggca tggcgaaaat 1560ccgtagatct gatgagacct gcggttcttt ttatatagag cgtaaataca ttcaatacct 1620tttaaagtat tctttgctgt attgatactt tgataccttg tctttcttac tttaatatga 1680cggtgatctt gctcaatgag gttattcaaa tatttcgatg tacaatgaca gtcaggttta 1740agtttaaaag ctttaattac tttagccatt gctaccttcg ttgaaggtgc ctgatctgta 1800attacctttt gaggtttacc aaattgttta atgagacgtt taataaacgc atatgctgaa 1860tgattatctc gttgcttacg caaccaaata tctaatgtat gtccctctgc atcaatggca 1920cgatataaat agctccattt tccttttatt ttgatgtacg tctcatcaat acgccatttg 1980taataagctt ttttatgctt tttcttccaa atttgatata aaattggggc atattcttga 2040acccaacggt agaccgttga atgatgaacg tttacaccac gtccccttaa tatttcagat 2100atatcacgat aactcaatgc atatcttaga tagtagccaa cggctacagt gat 2153642122DNAArtificial SequenceStaphylococcus aureus 64ggaaactaaa agagaaatat tggaagcaag ccatagcaga atatgaaaaa cgtttaggcc 60catacaccaa gatagacatc atagaagttc cagacgaaaa agcaccagaa aatatgagcg 120acaaagaaat tgagcaagta aaagaaaaag aaggccaacg aatactagcc aaaatcaaac 180cacaatcaac agtcattaca ttagaaatac aaggaaagat gctatcttcc gaaggattgg 240cccaagaatt gaaccaacgc atgacccaag ggcaaagcga ctttgtattc gtcattggcg 300gatcaaacgg cctgcacaag gacgtcttac aacgcagtaa ctacgcacta tcattcagca 360aaatgacatt cccacatcaa atgatgcggg ttgtgttaat tgaacaagtg tacagagcat 420ttaagattat gcgtggagaa gcgtaccaca aatgatgcgg ttttttatcc agttttttgt 480ttaatgaaca aggtaaatta cgagataata tttgaagaaa acaataaagt agagatggat 540ttccatatcc tctttagtag cggtttttat ctgtaaggtt tattaataat taaataaata 600ggcgggatag ttatatatag cttattaatg aaagaatatg attattaatt tagtattata 660ttttaatatt aaaaagaaga tatgaaataa ttattcatac cttccacctt acaataatta 720gttttcaatc gaatattaag attattagta gtcttaaaag ttaagacttc cttatattaa 780tgacctaatt tattatttgc ctcatgaatt atctttttat ttctttgata tgtcccaaac 840cacatcgtga tatacactac aataaatatt atgatgaaac taataatatt ctcaaagttc 900agatggaacc aacctgctag aatagcgagt gggaagaata ggattatcat caatataaag 960tgaactacag tctgttttgt tatactccaa tcggtatctg taaatatcaa attaccataa 1020gtaaacaaaa ttccaatcaa tgcccatagt gctacacata ttagcataat aaccgcttca 1080ttaaagtttt cataataaat tttacccata aaagaatctg gatatagtgg tacatattta 1140tcccttgaaa aaaataagtg aagtaatgac agaaatcata agaccagtga acgcaccttt 1200ttgaacagcg tggaataatt ttttcatagt gagatggacc attccatttg tttctaactt 1260caagtgatca atgtaattta gattgataat ttctgatttt gaaatacgca cgaatattga 1320accgacaagc tcttcaattt ggtaaagtcg ctgataaagt tttaaagctt tattattcat 1380tgttatcgca tacctgttta tcttctacta tgaactgtgc aatttgttct agatcaattg 1440ggtaaacatg atggttctgt tgcaaagtaa aaaaatatag ctaaccacta atttatcatg 1500tcagtgttcg cttaacttgc tagcatgatg ctaatttcgt ggcatggcga aaatccgtag 1560atctgatgag acctgcggtt ctttttatat agagcgtaaa tacattcaat accttttaaa 1620gtattctttg ctgtattgat actttgatac cttgtctttc ttactttaat atgacggtga 1680tcttgctcaa tgaggttatt cagatatttc gatgtacaat gacagtcagg tttaagttta 1740aaagctttaa ttactttagc cattgctacc ttcgttgaag gtgcctgatc tgtaattacc 1800ttttgaggtt taccaaattg tttaatgaga cgtttgataa acgcatatgc tgaatgatta 1860tctcgttgct tacgcaacca aatatctaat gtatgtccct ctgcatcaat ggcacgatat 1920aaatagctcc attttccttt tattttgatg tacgtctcat caatacgcca tttgtaataa 1980gcttttttat gctttttctt ccaaatttga tacaaaattg gggcatattc ttgaacccaa 2040cggtagaccg ttgaatgatg aacgtttaca ccacgttccc ttaatatttc agatatatca 2100cgataactca atgtatatct ta 212265737DNAArtificial SequenceStaphylococcus aureus 65tttaagatta tgcgtggaga agcatatcat aaatgatgcg gttatttcag ccgtaatttt 60ataatataaa gcagagttta ttaaatttta atgattactt tttattaaga attaattcta 120gttgatatat tataatgtga aacacaaaat aataatttgt aattgttagt ttataggcat 180ctgtatttgg aattttttgt agactattta aaaaatagtg tatataagta ttgagttcat 240gtattaactg tcttttttca tcgttcatca agtataagga tgtagagatt tgttggataa 300tttcttcgga tgtttttaaa attatcatta aattagatgg tatctgatct tgagttttgt 360ttttagtgta tgtatatttt aaaaaatttt tgattgttgt tatttgactc tcttttaatt 420tgacaccctc atcaataaat gtgttaaata tatcttcatt tgtacttaaa tcatcaaaat 480ttgccaacaa atatttgaac gtctctaaat cattatgttt gagttccgtt ttgctattcc 540ataattccaa accatttggt agaaagccca agctgtgatt ttgatctccc catatagctg 600aatttaaatc agtgagttga ttaatttttt caacacagaa atgtaatttt ggaatgagga 660atcgaagttg ttcttctact tgctgtactt ttcttttgtt ttcaataaaa tttctacacc 720atactgttat caaaccg 737661592DNAArtificial SequenceStaphylococcus aureus 66aactaaaaga gaaatattgg aagcaagcca tagcagaata tgaaaaacgt ttaggcccat 60acaccaagat agacatcata gaagttccag acgaaaaagc accagaaaat atgagtgaca 120aagaaattga gcaagtaaaa gaaaaagaag gccaacgaat actagccaaa atcaaaccac 180aatccacagt cattacatta gaaatacaag gaaagatgct atcttccgaa ggattggccc 240aagaattgaa ccaacgcatg acccaagggc aaagcgactt tgttttcgtc attggcggat 300caaacggcct gcacaaggac gtcttacaac gcagtaacta cgcactatca ttcagcaaaa 360tgacattccc acatcaaatg atgcgggttg tgttaattga acaagtgtac agagcattta 420agattatgcg aggagaagca tatcataaat gatgcggtta tttcagccgt aattttataa 480tataaagcag agtttattaa attttaatga ttacttttta ttaagaatta attctagttg 540atatattata atgtgaaaca caaaataata atttgtaatt gttagtttat aggcatctgt 600atttggaatt ttttgtagac tatttaaaaa atagtgtata taagtattga gttcatgtat 660taactgtctt ttttcatcgt tcatcaagta taaggatgta gagatttgtt ggataatttc 720ttcggatgtt tttaaaatta tcattaaatt agatggtatc tgatcttgag ttttgttttt 780agtgtatgta tattttaaaa aatttttgat tgttgttatt tgactctctt ttaatttgac 840accctcatca ataaatgtgt taaatatatc ttcatttgta cttaaatcat caaaatttgc 900caacaaatat ttgaacgtct ctaaatcatt atgtttgagt tccgttttgc tattccataa 960ttccaaacca tttggtagaa agcccaagct gtgattttga tctccccata tagctgaatt 1020taaatcagtg agttgattaa ttttttcaac acagaaatgt aattttggaa tgaggaatcg 1080aagttgttct tctacttgct gtacttttct tttgttttca ataaaatttc tacaccatac 1140tgttatcaaa ccgccaatta ttgtgcacaa tcctccaatg attgtagata aaattgacaa 1200tatattacac acctttctta gaggtttatt aacatctatt tttgaattta aaattattac 1260tttggtagcg ttataaccta tttaacagat tagagaaaaa ttgaatgatc gattgaagaa 1320tttccaaaat accgtcccat atgcgttgaa ggagatttct attttcttct gtattcaaat 1380ctttggcttt atcctttgct ttattcaata aatcatctga gtttttttca atatttttta 1440atacatcttt ggcattttgt ttaaatactt taggatcgga agttagggca ttagagtttg 1500ccacattaat catattatta ttaatcattt gaatttgatt atctgataat atctctgata 1560acctacgctc atcgaggact ttattaacag tg 159267730DNAArtificial SequenceStaphylococcus aureus 67agcatttaag attatgcgtg gagaagcata tcataaatga tgcggttatt tcagccgtaa 60ttttataata taaagcagag tttattaaat tttaatgatt actttttatt aagaattaat 120tctagttgat atattataat gtgaaacaca aaataataat ttgtaattgt tagtttatag 180gcatctgtat ttggaatttt ttgtagacta tttaaaaaat agtgtatata agtattgagt 240tcatgtatta actgtctttt ttcatcgttc atcaagtata aggatgtaga gatttgttgg 300ataatttctt cggatgtttt taaaattatc attaaattag atggtatctg atcttgagtt 360ttgtttttag tgtatgtata ttttaaaaaa tttttgattg ttgttatttg actctctttt 420aatttgacac cctcatcaat aaatgtgtta aatatatctt catttgtact taaatcatca 480aaatttgcca acaaatattt gaacgtctct aaatcattat gtttgagttc cgttttgcta 540ttccataatt ccaaaccatt tggtagaaag cccaagctgt gattttgatc tccccatata 600gctgaattta aatcagtgag ttgattaatt ttttcaacac agaaatgtaa ttttggaatg 660aggaatcgaa gttgttcttc tacttgctgt acttttcttt tgttttcaat aaaatttcta 720caccatactg 730681696DNAArtificial SequenceStaphylococcus aureus 68aaagagaaat attggaagca agccatagca gaatatgaaa aacgtttagg cccatacacc 60aagatagaca tcatagaagt tccagacgaa aaagcaccag aaaatatgag tgacaaagaa 120attgagcaag taaaagaaaa agaaggccaa cgaatactag ccaaaatcaa accacaatcc 180acagtcatta cattagaaat acaaggaaag atgctatctt ccgaaggatt ggcccaagaa 240ttgaaccaac gcatgaccca agggcaaagc gactttgttt tcgtcattgg cggatcaaac 300ggcctgcaca aggacgtctt acaacgcagt aactacgcac tatcattcag caaaatgaca 360ttcccacatc aaatgatgcg ggttgtgtta attgaacaag tgtacagagc atttaagatt 420atgcgaggag aagcatatca taaatgatgc ggttatttca gccgtaattt tataatataa 480agcagagttt attaaatttt aatgattact ttttattaag aattaattct agttgatata 540ttataatgtg aaacacaaaa taataatttg taattgttag tttataggca tctgtatttg 600gaattttttg tagactattt aaaaaatagt gtatataagt attgagttca tgtattaact 660gtcttttttc atcgttcatc aagtataagg atgtagagat ttgttggata atttcttcgg 720atgtttttaa aattatcatt aaattagatg gtatctgatc ttgagttttg tttttagtgt 780atgtatattt taaaaaattt ttgattgttg ttatttgact ctcttttaat ttgacaccct 840catcaataaa tgtgttaaat atatcttcat ttgtacttaa atcatcaaaa tttgccaaca 900aatatttgaa cgtctctaaa tcattatgtt tgagttccgt tttgctattc cataattcca 960aaccatttgg tagaaagccc aagctgtgat tttgatctcc ccatatagct gaatttaaat 1020cagtgagttg attaattttt tcaacacaga aatgtaattt tggaatgagg aatcgaagtt 1080gttcttctac ttgctgtact tttcttttgt tttcaataaa atttctacac catactgtta 1140tcaaaccgcc aattattgtg cacaatcctc caatgattgt agataaaatt gacaatatat 1200tacacacctt tcttagaggt ttattaacat ctatttttga atttaaaatt attactttgg 1260tagcgttata acctatttaa cagattagag

aaaaattgaa tgatcgattg aagaatttcc 1320aaaataccgt cccatatgcg ttgaaggaga tttctatttt cttctgtatt caaatctttg 1380gctttatcct ttgctttatt caataaatca tctgagtttt tttcaatatt ttttaataca 1440tctttggcat tttgtttaaa tactttagga tcggaagtta gggcattaga gtttgccaca 1500ttaatcatat tattattaat catttgaatt tgattatctg ataatatctc tgataaccta 1560cgctcatcga ggactttatt aacagtgtct tcaacttgtt gttgtgtgat ttgtttatct 1620tgattttgtt taatatctgc aagttgttct ttaatatctg ctatagaagc atttaaagct 1680tcatctgaat acccat 169669991DNAArtificial SequenceStaphylococcus aureus 69accattttag ctgtagggaa actaaaagag aaatactgga agcaagccat agcagaatat 60gaaaaacgtt taggcccata caccaagata gacatcatag aagttccaga cgaaaaagca 120ccagaaaata tgaactacaa agaaattgag caagtaaaag aaaaagaagg ccaacgaata 180ctagccaaaa tcaaaccaca atcaacagtc attacattag aaatacaagg aaagatgcta 240tcttccgaag gattggccca agaattgaac caacgcatga cccaagggca aagcgacttt 300gtattcgtca ttggcggatc aaacggcctg cacaaggacg tcttacaacg cagtaactac 360gcactatcat tcagcaaaat gacattccca catcaaatga tgcgggttgt gttaattgaa 420caagtgtaca gagcatttaa gattatgcga ggagaagcgt atcataagtg atggtaaaaa 480atatgagtaa gtagatgaag agtgaaaatc agattaatta ataataatgt atcaaattta 540aataaagggg tttttaagta tgaatttaag aggtcatgaa aatagactta aatttcatgc 600gaaatatgat gtgacaccta tatcacattt aaaattatta gaaggtcaaa agaaagacgg 660tgaaggcggc atactgacag atagctatta ctgtttttca tacagcttaa aaggtaattc 720taaaaaagtt ttaggtacgt ttaattgtgg ttatcatatt gctgaagatt tactaaaatt 780atcaaatcaa gataaattac ctttatttaa cccgtttaaa gtaattaatg aaggtaatca 840attgcagggc gtaacgaata aaggtaattt aaatattaat aggcaaagaa aacagtataa 900tgaagtggct ttacagcttt caaatgctat taatttaatc ataatttgtt atgaggataa 960tattaaagaa ccactttcaa cgataaaata c 991701282DNAArtificial SequenceStaphylococcus aureus 70accattttag ctgtagggaa actaaaagag aaatattgga agcaagccat agcagaatat 60gaaaaacgtt taggcccata caccaagata gacatcatag aagttccaga cgaaaaagca 120ccagaaaata tgagcgacaa agaaattgag caagtaaaag aaaaagaagg ccaacgaata 180ctagccaaaa tcaaaccaca atccacagtc attacattag aaatacaagg aaagatgcta 240tcttccgaag gattggccca agaattgaac caacgcatga cccaagggca aagcgacttt 300gtattcgtca ttggcggatc aaacggcctg cacaaggacg tcttacaacg cagtaactat 360gcactatcat ttagcaaaat gacattccca catcaaatga tgcgggttgt gttaattgaa 420caagtgtata gagcatttaa gattatgcgt ggagaagcgt accacaaata aaactaaaaa 480atatgagaaa attattaaat tagctcaaat ctttgaagaa taaaaagtga atattaagtt 540tgataattta ggtacaagta aagattaaga atttccatta tttaatacat ggtgtgtaaa 600tcgacttctt tttgtattag atgtttgcag taagcgatgt aaagaagatg ctaataaata 660tgtgaggaat gattacgata ctagataagc ggctaatgaa attttttaaa gtacatatat 720agacatattt ttcatttagt aaaattttga atttcacttt gctaagacta gtgtctagaa 780atttataatg atttattaac acctatttga aacttaagta taataaatga ttcggatttt 840atttttaata aagacaaact tgaacgtagc aaagtagttt ttatgataaa taataagttt 900taataatgtg acgcttttat ataagcacat tattatgaac aatgtgaatt gagcatctac 960aattacatta ataaatatat aaatgatgat ttaaattcac atatatttat aatacacata 1020ctatatgaaa gttttgatta tccgaataaa tgctaaaatt aataaaataa ttaaaggaat 1080catacttatt atacgtatac gtttagctac tgaactactg gattcatttg gagattctag 1140tagttctttt tcaatctcta aatctaaatc agttttgtaa taaccattaa ttcctaatct 1200ttcatctagc tctgtacttt tttcatcatt tttatctttg ttgatatgtt ccattttctc 1260gcctcttttt aatcaagtag aa 1282711108DNAArtificial SequenceStaphylococcus aureus 71accattttag ctgtagggaa actaaaagag aaatattgga agcaagccat agcagaatat 60gaaaaacgtt taggcccata caccaagata gacatcatag aagttccaga cgaaaaagca 120ccagaaaata tgagcgacaa agaaattgag caagtaaaag aaaaagaagg ccaacgaata 180ctagccaaaa tcaaaccaca atccacagtc attacattag aaatacaagg aaagatgcta 240tcttccgaag gattggccca agaattgaac caacgcatga cccaagggca aagcgacttt 300gtattcgtca ttggcggatc aaacggcctg cacaaggacg tcttacaacg cagtaactat 360gcactatcat ttagcaaaat gacattccca catcaaatga tgcgggttgt gttaattgaa 420caagtgtata gagcatttaa gattatgcgt ggagaagcgt accacaaata aaactaaaaa 480atatgagaaa attattaaat tagctcaaat ctttgaagaa taaaaagtga atattaagtt 540tgataattta ggtacaagta aagattaaga atttccatta tttaatacat ggtgtgtaaa 600tcgacttctt tttgtattag atgtttgcag taagcgatgt aaagaagatg ctaataaata 660tgtgaggaat gattacgata ctagataagc ggctaatgaa attttttaaa gtacatatat 720agacatattt ttcatttagt aaaattttga atttcacttt gctaagacta gtgtctagaa 780atttataatg atttattaac acctatttga aacttaagta taataaatga ttcggatttt 840atttttaata aagacaaact tgaacgtagc aaagtagttt ttatgataaa taataagttt 900taataatgtg acgcttttat ataagcacat tattatgaac aatgtgaatt gagcatctac 960aattacatta ataaatatat aaatgatgat ttaaattcac atatatttat aatacacata 1020ctatatgaaa gttttgatta tccgaataaa tgctaaaatt aataaaataa ttaaaggaat 1080catacttatt atacgtatac gtttagct 1108721530DNAArtificial SequenceStaphylococcus aureus 72ttagctgtag ggaaactaaa agagaaatat tggaagcaag ccatagcaga atatgaaaaa 60cgtttaggcc catacaccaa gatagacatc atagaagttc cagacgaaaa agcaccagaa 120aatatgagcg acaaagaaat tgagcaagta aaagaaaaag aaggccaacg aatactagcc 180aaaatcaaac cacaatccac agtcattaca ttagaaatac aaggaaagat gctatcttcc 240gaaggattgg cccaagaatt gaaccaacgc atgacccaag ggcaaagcga ctttgtattc 300gtcattggcg gatcaaacgg cctgcacaag gacgtcttac aacgcagtaa ctatgcacta 360tcatttagca aaatgacatt cccacatcaa atgatgcggg ttgtgttaat tgaacaagtg 420tatagagcat ttaagattat gcgtggagaa gcatatcata aatgatgcgg ttttttcagc 480cgcttcataa aggggggtga tcatatcgga acgtatgagg tttatgagaa ttgctgctat 540gtttttatga agcgtatcat aaatgatgca gtttttgata attttttctt tatcagagat 600tttactaaaa atcccctcaa agtttgtttt tttcaacttc aactttgaag ggaataaata 660aggaacttat ttatatttat cctttatctc attaatatct atttttttat taataatatt 720ataaatatta aattctttag aaaagtcact atcactctta ttcttcatac taaacgttat 780taatctaata atatcagcta ctatttcttt aaattctatt gcatcttctt ttttataagt 840agcgcctgta tgaacaattt tatttctcat accatagtaa tctttcatat atttttttac 900acaattttta atttcattag aattatccaa atctagatta tcaattgtct ttaataaatg 960atcattaaca acattagcat acccacatcc aagcttcttt tttatctctt catcacttaa 1020attttcatct aatttataat atctttctaa aaaatttgtg ataaaaactt ctaatgcagt 1080ctgaatttgt acaattgcta aattatagtc agatttataa aaagaacgtt caccttttct 1140catagccaaa acataaatat tgctaggatg attattgaaa atattataat tttttttaat 1200atttaataaa tcactttttt tgatagatga atactgatct tcttctatct ttccaggcat 1260gtcaatcatg aaaatactca tctcttttat atttccatct atagtatata ttatataata 1320tggaatactt aatatatccc ctaatgatag ctggtatata ttatgatact gatatttaac 1380gctaataatt ttaataagat tatttagaca attaaattgc ttattaaaaa ttttcgttag 1440actattactt ttctttgatt ccctagaagt agaatttgat ttcaattttt taaactgatt 1500gtgcttgatt attgaagtta tttcaacata 1530731256DNAArtificial SequenceStaphylococcus aureus 73gctgtaggga aactaaaaga gaaatattgg aagcaagcca tagcagaata tgaaaaacgt 60ttaggcccat acaccaagat agacatcata gaagttccag acgaaaaagc accagaaaat 120atgagcgaca aagaaattga gcaagtaaaa gaaaaagaag gccaacgaat actagccaaa 180attaaaccac aatccacagt cattacatta gaaatacaag gaaagatgct atcttccgaa 240ggattggccc aagaattgaa ccaacgcatg acccaagggc aaagcgactt tgtattcgtc 300attggcggat caaacggcct gcacaaggac gtcttacaac gcagtaacta cgcactatca 360ttcagcaaaa tgacattccc acatcaaatg atgcgggttg tgttaattga gcaagtgtat 420agagcattta agattatgcg tggagaagca tatcataaat gatgcggttt tttcagccgc 480ttcataaagg gattttgaat gtatcagaac atatgaggtt tatgtgaatt gctgttatgt 540ttttaagaag catatcataa gtgatgcggt ttttattaat tagttgctaa aaaatgaagt 600atgcaatatt aattattatt aaattttgat atatttaaag aaagattaag tttagggtga 660atgaatggct tatcaaagtg aatatgcatt agaaaatgaa gtacttcaac aacttgagga 720attgaactat gaaagagtaa atatacataa tattaaatta gaaattaatg aatatctcaa 780agaactagga gtgttgaaaa atgaataagc agacaaatac tccagaacta agatttccag 840agtttgatga ggaatggaaa aaaaggaaat taggtgaagt agtaaattat aaaaatggtg 900gttcatttga aagtttagtg aaaaaccatg gtgtatataa actcataact cttaaatctg 960ttaatacaga aggaaagttg tgtaattctg gaaaatatat cgatgataaa tgtgttgaaa 1020cattgtgtaa tgatacttta gtaatgatac tgagcgagca agcaccagga ctagttggaa 1080tgactgcaat tatacctaat aataatgagt atgtactaaa tcaacgagta gcagcactag 1140tgcctaaaca atttatagat agtcaatttc tatctaagtt aattaataga aaccagaaat 1200atttcagtgt gagatctgct ggaacaaaag tgaaaaatat ttctaaagga catgta 1256741032DNAArtificial SequenceStaphylococcus aureus 74accattttag ctgtagggaa actaaaagag aaatattgga agcaagccat agcagaatat 60gaaaaacgtt taggcccata caccaagata gacatcatag aagttccaga cgaaaaagca 120ccagaaaata tgagcgacaa agaaattgag caagtaaaag aaaaagaagg ccaacgaata 180ctagccaaaa ttaaaccaca atccacagtc attacattag aaatacaagg aaagatgcta 240tcttccgaag gattggccca agaattgaac caacgcatga cccaagggca aagcgacttt 300gtattcgtca ttggcggatc aaacggcctg cacaaggacg tcttacaacg cagtaactac 360gcactatcat tcagcaaaat gacattccca catcaaatga tgcgggttgt gttaattgag 420caagtgtata gagcatttaa gattatgcgt ggagaagcat atcataagtg atgcggtttt 480tattaattag ttgctaaaaa atgaagtatg caatattaat tattattaaa ttttgatata 540tttaaagaaa gattaagttt agggtgaatg aatggcttat caaagtgaat atgcattaga 600aaatgaagta cttcaacaac ttgaggaatt gaactatgaa agagtaaata tacataatat 660taaattagaa attaatgaat atctcaaaga actaggagtg ttgaaaaatg aataagcaga 720caaatactcc agaactaaga tttccagagt ttgatgagga atggaaaaaa aggaaattag 780gtgaagtagt aaattataaa aatggtggtt catttgaaag tttagtgaaa aaccatggtg 840tatataaact cataactctt aaatctgtta atacagaagg aaagttgtgt aattctggaa 900aatatatcga tgataaatgt gttgaaacat tgtgtaatga tactttagta atgatactga 960gcgagcaagc accaggacta gttggaatga ctgcaattat acctaataat aatgagtatg 1020tactaaatca ac 1032751116DNAArtificial SequenceStaphylococcus aureus 75accattttag ctgtagggaa actaaaagag aaatattgga agcaagccat agcagaatat 60gaaaaacgtt taggcccata caccaagata gacatcatag aagttccaga cgaaaaagca 120ccagaaaata tgagcgacaa agaaattgag caagtaaaag aaaaagaagg ccaacgaata 180ctagccaaaa ttaaaccaca atccacagtc attacattag aaatacaagg aaagatgcta 240tcttccgaag gattggccca agaattgaac caacgcatga cccaagggca aagcgacttt 300gtattcgtca ttggcggatc aaacggcctg cacaaggacg tcttacaacg cagtaactac 360gcactatcat tcagcaaaat gacattccca catcaaatga tgcgggttgt gttaattgag 420caagtgtata gagcatttaa gattatgcgt ggagaagcat atcataaatg atgcggtttt 480ttcagccgct tcataaaggg attttgaatg tatcagaaca tatgaggttt atgtgaattg 540ctgttatgtt tttaagaagc atatcataaa tgatgcggtt ttttcagccg cttcataaag 600ggattttgaa tgtatcagaa catatgaggt ttatgtgaat tgctgttatg tttttaagaa 660gcatatcata agtgatgcgg tttttattaa ttagttgcta aaaaatgaag tatgcaatat 720taattattat taaattttga tatatttaaa gaaagattaa gtttagggtg aatgaatggc 780ttatcaaagt gaatatgcat tagaaaatga agtacttcaa caacttgagg aattgaacta 840tgaaagagta aatatacata atattaaatt agaaattaat gaatatctca aagaactagg 900agtgttgaaa aatgaataag cagacaaata ctccagaact aagatttcca gagtttgatg 960aggaatggaa aaaaaggaaa ttaggtgaag tagtaaatta taaaaatggt ggttcatttg 1020aaagtttagt gaaaaaccat ggtgtatata aactcataac tcttaaatct gttaatacag 1080aaggaaagtt gtgtaattct ggaaaatata tcgatg 1116761100DNAArtificial SequenceStaphylococcus aureus 76accattttag ctgtagggaa actaaaagag aaatattgga agcaagccat agcagaatat 60gaaaaacgtt taggcccata caccaagata gacatcatag aagttccaga cgaaaaagca 120ccagaaaata tgagcgacaa agaaattgag caagtaaaag aaaaagaagg ccaacgaata 180ctagccaaaa ttaaaccaca atccacagtc attacattag aaatacaagg aaagatgcta 240tcttccgaag gattggccca agaattgaac caacgcatga cccaagggca aagcgacttt 300gtattcgtca ttggcggatc aaacggcctg cacaaggacg tcttacaacg cagtaactac 360gcactatcat tcagcaaaat gacattccca catcaaatga tgcgggttgt gttaattgag 420caagtgtata gagcatttaa gattatgcgt ggagaagcgt atcacaaata aaactaaaaa 480ataagttgta tataacttat tttgaaattg gttaagtata taatatctcc aataaaatgt 540agttaactta cgataatgct gaactatagc tttgtaaact aaaatgtaaa taattacaat 600caaattgcaa caatatagtt caagaatgct acaatttgag gacagattga tagcattaat 660ccctttaaaa tgaagctagg agataactta cattatgatt agtaaacaaa taaaggattt 720acgaaagcaa cataattata ctcaagaaga gctagctgaa aaattaaata cttcaagaca 780aacaatttct aaatgggaac aaggtatttc agaaccagac ttaattatgc ttatgcaatt 840gtcacaatta ttttctgtta gtacagacta tctcattaca ggaagtgaca atattattaa 900aaaagataat aaaagctatt atgaaatgaa tttttgggca tttatgtctg aaaaatggtg 960ggtaattatt attatagtaa tcataatttg tggaacaata ggacaaattt tttcaaacta 1020atgtaagtat ctctcaaata ttttgggagg ttttattatg aaaatcaaaa aattattaaa 1080gacattatta attattttat 1100771118DNAArtificial SequenceStaphylococcus aureus 77atgaaaatca ccattttagc tgtagggaaa ctaaaagaga aatattggaa gcaagccata 60gcagaatatg aaaaacgttt aggcccatac accaagatag acatcataga agttccagac 120gaaaaagcac cagaaaatat gagcgacaaa gaaattgagc aagtaaaaga aaaagaaggc 180caacgaatac tagccaaaat caaaccacaa tcaacagtca ttacattaga aatacaagga 240aagatgctat cttccgaagg attggcccaa gaattgaacc aacgcatgac ccaagggcaa 300agcgactttg tattcgtcat tggcggatca aacggcctgc acaaggacgt cttacaacgc 360agtaactacg cactatcatt cagcaaaatg acattcccac atcaaatgat gcgggttgtg 420ttaattgaac aagtgtacag agcatttaag attatgcgtg gagaagcgta tcacaaataa 480aactaaaaaa taagttgtat ataacttatt ttgaaattgg ttaagtatat agtatctcca 540ataaaatgta gttaacttac gataatgctg aactatagct ttgtaaacta aaatgtaaat 600aattacaatc aaattgcaac aatatagttc aagaatgcta caatttgagg acagattgat 660agcattaatc cctttaaaat gaagctagga gataacttac attatgatta gtaaacaaat 720aaaggattta cgaaagcaac ataattatac tcaagaagag ctagctgaaa aattaaatac 780ttcaagacaa acaatttcta aatgggaaca aggtatttca gaaccagact taattatgct 840tatgcaattg tcacaattat tttctgttag tacagactat ctcattacag gaagtgacaa 900tattattaaa aaagataata aaagctatta tgaaatgaat ttttgggcat ttatgtctga 960aaaatggtgg gtaattatta ttatagtaat cataatttgt ggaacaatag gacaaatttt 1020ttcaaactaa tgtaagtatc tctcaaatat tttgggaggt tttattatga aaatcaaaaa 1080attattaaag acattattaa ttattttatt atgttttg 1118781168DNAArtificial SequenceStaphylococcus aureus 78atgaaaatca ccattttagc tgtagggaaa ctaaaagaga aatattggaa gcaagccata 60gcagaatatg aaaaacgttt aggcccatac accaagatag acatcataga agttccagac 120gaaaaagcac cagaaaatat gagcgacaaa gaaattgagc aagtaaaaga aaaagaaggc 180caacgaatac tagccaaaat caaaccacaa tcaacagtca ttacattaga aatacaagga 240aagatgctat cttccgaagg attggcccaa gaattgaacc aacgcatgac ccaagggcaa 300agcgactttg tattcgtcat tggcggatca aacggcctgc acaaggacgt cttacaacgc 360agtaactacg cactatcatt cagcaaaatg acattcccac atcaaatgat gcgggttgtg 420ttaattgaac aagtgtacag agcatttaag attatgcgtg gagaagcgta tcacaaataa 480aactaaaaaa taagttgtat ataacttatt ttgaaattgg ttaagtatat agtatctcca 540ataaaatgta gttaacttac gataatgctg aactatagct ttgtaaacta aaatgtaaat 600aattacaatc aaattgcaac aatatagttc aagaatgcta caatttgagg acagattgat 660agcattaatc cctttaaaat gaagctagga gataacttac attatgatta gtaaacaaat 720aaaggattta cgaaagcaac ataattatac tcaagaagag ctagctgaaa aattaaatac 780ttcaagacaa acaatttcta aatgggaaca aggtatttca gaaccagact taattatgct 840tatgcaattg tcacaattat tttctgttag tacagactat ctcattacag gaagtgacaa 900tattattaaa aaagataata aaagctatta tgaaatgaat ttttgggcat ttatgtctga 960aaaatggtgg gtaattatta ttatagtaat cataatttgt ggaacaatag gacaaatttt 1020ttcaaactaa tgtaagtatc tctcaaatat tttgggaggt tttattatga aaatcaaaaa 1080attattaaag acattattaa ttattttatt atgttttgta ttgtctgtta ttgtgcaaaa 1140tatttcaatg ctatggcata ttgtgagc 1168791134DNAArtificial SequenceStaphylococcus aureus 79atgaaaatca ccattttagc tgtagggaaa ctaaaagaga aatattggaa gcaagccata 60gcagaatatg aaaaacgttt aggcccatac accaagatag acatcataga agttccagac 120gaaaaagcac cagaaaatat gagcgacaaa gaaattgagc aagtaaaaga aaaagaaggc 180caacgaatac tagccaaaat caaaccacaa tccacagtca ttacattaga aatacaagga 240aagatgctat cttccgaagg attggcccaa gaattgaacc aacgcatgac ccaagggcaa 300agcgactttg tattcgtcat tggcggatca aacggcctgc acaaggacgt cttacaacgc 360agtaactatg cactatcatt tagcaaaatg acattcccac atcaaatgat gcgggttgtg 420ttaattgaac aagtgtatag agcatttaag attatgcgtg gagaggcgta tcataaataa 480aactaaaaaa cggattgtgt ataatatatt ttaaatataa aaaggattga ttttatgtta 540aataaattag aaaatgttag ttataaatca ttcgataatt acactagtga agatgatttg 600actaaagtaa atatattttt tggaagaaat gggagtggaa aaagctcatt aagtgaatgg 660ttaagaagac tagataatga aaaaagtgtt atctttaata ctggttactt aaaaaataat 720attgaagaag ttgaagaaat agatggtgtg aatttggtta ttggagaaga atctataaat 780catagtgacc aaattaagca tttaaatagc gctataaata gtttagaaaa ttttattact 840cggaaaaata gtgaacttaa gcattcaaaa gaaagaattt acaataaaat gaatatcaga 900ctaaatgaag ctagagaaag atttgaaata ggtagtaatg tggttaagca gaagaggaat 960gctgacaaag atccagttaa tgctttttat agttggaaga aaaatgctaa cgatataatt 1020caagagatga ctattgaatc tttagatgaa ttagaagaaa gaataacaag aaaagaagtc 1080ttattaaata atataaaaac accaatttta gcttttgatt ataatgattt tagt 113480818DNAArtificial SequenceStaphylococcus aureus 80aatattggaa gcaagccata gcagaatatg aaaaacgttt aggcccatac accaagatag 60acatcataga agttccagac gaaaaagcac cagaaaatat gagcgacaaa gaaattgagc 120aagtgtatag agcatttaag attatgcgtg gagaagcata tcataaatga tgcggttttt 180tcagccgctt cataaaggga ttttgaatgt atcagaacat atgaggttta tgtgaattgc 240tgttatgttt ttaagaagct tatcataagt aatgaggttc atgatttttg acatagttag 300cctccgcagt ctttcatttc aagtaaataa tagcgaaata ttctttatac tgaatactta 360tagtgaagca aagttctagc tttgagaaaa ttctttctgc aactaaatat agtaaattac 420ggtaaaatat aaataagtac atattgaaga aaatgagaca taatatattt tataatagga 480gggaatttca aatgatagac aactttatgc aggtccttaa attaattaaa gagaaacgta 540ccaataatgt agttaaaaaa tctgattggg ataaaggtga tctatataaa actttagtcc 600atgataagtt acccaagcag ttaaaagtgc atataaaaga agataaatat tcagttgtag 660ggaaggttgc tactgggaac tatagtaaag ttccttggat ttcaatatat gatgagaata 720taacaaaaga aacaaaggat ggatattatt

tggtatatct ttttcatccg gaaggagaag 780gcatatactt atcttgaatc aaggatggtc aaagataa 818811090DNAArtificial SequenceStaphylococcus aureus 81atgaaaatca ccattttagc tgtagggaaa ctaaaagaga aatattggaa gcaagccata 60gcagaatatg aaaaacgttt aggcccatac accaagatag acatcataga agttccagac 120gaaaaagcac cagaaaatat gagcgacaaa gaaattgagc aagtaaaaga aaaagaaggc 180caacgaatac tagccaaaat caaaccacaa tcaacagtca ttacattaga aatacaagga 240aagatgctat cttccgaagg attggcccaa gaattgaacc aacgcatgac ccaagggcaa 300agcgactttg tattcgtcat tggcggatca aacggcctgc acaaggacgt cttacaacgc 360agtaactacg cactatcatt cagcaaaatg acattcccac atcaaatgat gcgggttgtg 420ttaattgaac aagtgtacag agcatttaag attatgcgtg gagaagcata tcataagtga 480tgcggttttt attaattagt tgctaaaaaa tgaagtatgc aatattaatt attattaaat 540tttgatatat ttaaagaaag attaagttta gggtgaatga atggcttatc aaagtgaata 600tgcattagaa aatgaagtac ttcaacaact tgaggaattg aactatgaaa gagtaaatat 660acataatatt aaattagaaa ttaatgaata tctcaaagaa ctaggagtgt tgaaaaatga 720ataagcagac aaatactcca gaactaagat ttccagagtt tgatgaggaa tggaaaaaaa 780ggaaattagg tgaagtagta aattataaaa atggtggttc atttgaaagt ttagtgaaaa 840accatggtgt atataaactc ataactctta aatctgttaa tacagaagga aagttgtgta 900attctggaaa atatatcgat gataaatgtg ttgaaacatt gtgtaatgat actttagtaa 960tgatactgag cgagcaagca ccaggactag ttggaatgac tgcaattata cctaataata 1020atgagtatgt actaaatcaa cgagtagcag cactagtgcc taaacaattt atagatagtc 1080aatttctatc 1090821063DNAArtificial SequenceStaphylococcus aureus 82atgaaaatca ccattttagc tgtagggaaa ctaaaagaga aatattggaa gcaagccata 60gcagaatatg aaaaacgttt aggcccatac accaagatag acatcataga agttccagac 120gaaaaagcac cagaaaatat gagcgacaaa gaaattgagc aagtaaaaga aaaagaaggc 180caacgaatac tagccaaaat caaaccacaa tcaacagtca ttacattaga aatacaagga 240aagatgctat cttccgaagg attggcccaa gaattgaacc aacgcatgac ccaagggcaa 300agcgactttg tattcgtcat tggcggatca aacggcctgc acaaggacgt cttacaacgc 360agtaactacg cactatcatt cagcaaaatg acattcccac atcaaatgat gcgggttgtg 420ttaattgaac aagtgtacag agcatttaag attatgcgtg gagaagcata tcataagtga 480tgcggttttt attaattagt tgctaaaaaa tgaagtatgc aatattaatt attattaaat 540tttgatatat ttaaagaaag attaagttta gggtgaatga atggcttatc aaagtgaata 600tgcattagaa aatgaagtac ttcaacaact tgaggaattg aactatgaaa gagtaaatat 660acataatatt aaattagaaa ttaatgaata tctcaaagaa ctaggagtgt tgaaaaatga 720ataagcagac aaatactcca gaactaagat ttccagagtt tgatgaggaa tggaaaaaaa 780ggaaattagg tgaagtagta aattataaaa atggtggttc atttgaaagt ttagtgaaaa 840accatggtgt atataaactc ataactctta aatctgttaa tacagaagga aagttgtgta 900attctggaaa atatatcgat gataaatgtg ttgaaacatt gtgtaatgat actttagtaa 960tgatactgag cgagcaagca ccaggactag ttggaatgac tgcaattata cctaataata 1020atgagtatgt actaaatcaa cgagtagcag cactagtgcc taa 106383756DNAArtificial SequenceStaphylococcus aureus 83tgacattccc acatcaaatg atgcgggttg tgttaattga gcaagtgtat agagcattta 60agattatgcg tggagaagcg tatcacaaat aaaactaaaa aataggttgc gcataatata 120attagaaagg aattagacat aaattaggag tccttcacag aatagcgaag gactcccatt 180aaatatatta tggtgtaaag aaatcacaaa tcaatatata tacttaatac catatattaa 240cttgtactat tataaagtac gacatcagta ttaggtatca ctttgaacac atgaatttca 300ttatcacttt tattattcac aaaaaatttt ccaattctca attactgaat tatgtgtata 360catgttgtta aaaattaata aaggatattt atgtttgttt aaagcatatc acaagtgatg 420cggtttttta taaagattta cttgttagtg attttgataa aaatgcttaa tactatttca 480ataatatgta tttaaaaatt agattaatag tatttaactt caaatggcct cgtataaact 540catagcaaat taacgtaaat caatgaaata aaatgaaaac aatttcaaga atacattata 600aacataaagt atacaaaaaa taaatgagcg tatttgttta aacgtataca ctcattttta 660ttaaattaat ttattatatt ttacgattgt tatttatgaa attaacaaat tccatttttg 720atagtgaaat taaaagcttt atcacttatt attgat 75684771DNAArtificial SequenceStaphylococcus aureus 84tgacattccc acatcaaatg atgcgggttg tgttaattga gcaagtgtat agagcattta 60agattatgcg tggagaagcg tatcacaaat aaaactaaaa aataggttgc gcataatata 120attagaaagg aattagacat aaattaggag tccttcacag aatagcgaag gactcccatt 180aaatatatta tggtgtaaag aaatcacaaa tcaatatata tacttaatac catatattaa 240cttgtactat tataaagtac gacatcagta ttaggtatca ctttgaacac atgaatttca 300ttatcacttt tattattcac aaaaaatttt ccaattctca attactgaat tatgtgtata 360catgttgtta aaaattaata aaggatattt atgtttgttt aaagcatatc acaagtgatg 420cggtttttta taaagattta cttgttagtg attttgataa aaatgcttaa tactatttca 480ataatatgta tttaaaaatt agattaatag tatttaactt caaatggcct cgtataaact 540catagcaaat taacgtaaat caatgaaata aaatgaaaac aatttcaaga atacattata 600aacataaagt atacaaaaaa taaatgagcg tatttgttta aacgtataca ctcattttta 660ttaaattaat ttattatatt ttacgattgt tatttatgaa attaacaaat tccatttttg 720atagtgaaat taaaagcttt atcacttatt attgataatt ttgactgcat c 77185681DNAArtificial SequenceStaphylococcus aureus 85ttcagcaaaa tgacattccc acatcaaatg atgcgggttg tgttaattga acaagtgtac 60agagcattta agattatgcg tggagaagcg tatcataagt agcggaggag ttttttacct 120tgtgacttat cataaagtac gatgtttatg taagtgatta tcattattta agcaggtttt 180tcaaattaaa taataacaag aataaaatgc acttagcgac attgaaattt attaatctag 240taaactaata gatttataga aaattttatt tgcaagggga taattttgaa aagtagtatt 300ttctatcttt ccataataca ttgtaattac aacggagggg atattgtgat gaagtgtata 360gataaaacgt gggttagcta ttataaagaa ttagctgata agttaacaga ttatcaaaat 420aaacgttatg aattaattag aaatagtgaa ggaagtatat aaaaaaacgg gaataaaatt 480ccctacttta gcaagtgata atgtattgat ggacatagat ccttttacaa tatttgcatt 540atttaataaa aattccatga gagaaactaa taaggtaaaa atattaacag aattagcttc 600ggaattgaat attaagtcca aaattccgtc agtttttgac agtattccaa cagtcaataa 660tctgaatgct acatattata a 681861119DNAArtificial SequenceStaphylococcus aureus 86gacattccca catcaaatga tgcgggttgt gttaattgag caagtgtata gagcatttaa 60gattatgcgt ggagaggcgt atcataagta aaactaaaaa attctgtatg aggagataat 120aatttggagg gtgttaaatg gtggacatta aatccacgtt cattcaatat ataagatata 180tcacgataat tgcgcatata acttaagtag tagctaacag ttgaaattag gccctatcaa 240attggtttat atctaaaatg attaatatag aatgcttctt tttgtcctta ttaaattata 300aaagtaactt tgcaatagaa acagttattt cataatcaac agtcattgac gtagctaagt 360aatgataaat aatcataaat aaaattacag atattgacaa aaaatagtaa atataccaat 420gaagtttcaa aagaacaatt ccaagaaatt gagaatgtaa ataataaggt caaagaattt 480tattaagatt tgaaagagta tcaatcaaga aagatgtagt tttttaataa actatttgga 540aaataattat cataatttaa aaactgacaa tttgcgagac tcataaaatg taataatgga 600aatagatgta aaatataatt aaggggtgta atatgaagat taatatttat aaatctattt 660ataattttca ggaaacaaat acaaattttt tagagaatct agaatcttta aatgatgaca 720attatgaact gcttaatgat aaagaacttg ttagtgattc aaatgaatta aaattaatta 780gtaaagttta tatacgtaaa aaagacaaaa aactattaga ttggcaatta ttaataaaga 840atgtatacct agatactgaa gaagatgaca atttattttc agaatccggt catcattttg 900atgcaatatt atttctcaaa gaagatacta cattacaaaa taatgtatat attatacctt 960ttggacaagc atatcatgat ataaataatt tgattgatta tgacttcgga attgattttg 1020cagaaagagc aatcaaaaat gaagacatag ttaataaaaa tgttaatttt tttcaacaaa 1080acaggcttaa agagattgtt aattatagaa ggaatagtg 1119871073DNAArtificial SequenceStaphylococcus aureus 87tattggaagc aagccatagc agaatatgaa aaacgtttag gcccatacac caagatagac 60atcatagaag ttccagacga aaaagcacca gaaaatatga gcgacaaaga aattgagcaa 120gtaaaagaaa aagaaggcca acgaatacta gccaaaatca aaccacaatc cacagtcatt 180acattagaaa tacaaggaaa gatgctatct tccgaaggat tggcccaaga attgaaccaa 240cgcatgaccc aagggcaaag cgactttgta ttcgtcattg gcggatcaaa cggcctgcac 300aaggacgtct tacaacgcag taactatgca ctatcattta gcaaaatgac attcccacat 360caaatgatgc gggttgtgtt aattgaacaa gtgtatagag catttaagat tatgcgtgga 420gaggcgtatc ataagtgatg cttgttagaa tgatttttaa caatatgaaa tagctgtgga 480agcttaaaca atttgtttat ctaagtactt atttaataat tgattgaact gtgattggca 540ccaggctgtc tggtaaattg agaagttggg ttttggagcg tataaatgat agaattaata 600taaaattcaa tttgaggagt aggagattat gtcgaatata aaaacaacac tagagacgtc 660cgtaggacta gaaaaagaca acgataagct atttgattat ataactgaat tagagattca 720aaacacgcct gaaaaccggg aagcaaaagt tgttattgaa gaaaggttac ataaagaata 780taaatatgaa ttagatcaaa tgacaccaga gtatggaata caaaaaggca gtgttagaat 840aggtcatgca gatgttgtaa tatttcatga ttctaaagat aaatctcaag agaatattaa 900aataatagta gagtgtaaaa gaaagaatcg cagggatggt attgaacaat taaaaacata 960tcttgcaggg tgtgagtctg cagaatacgg cgtttggttt aatggagaag atatagtata 1020tataaaacga ttgaaaaaag caccacattg gaaaacagta tttaatatac cga 107388595DNAArtificial SequenceStaphylococcus aureus 88atgaaaatca ccattttagc tgtagggaaa ctaaaagaga aatattggaa gcaagccata 60gcagaatatg aaaaacgttt aggcccatac accaagatag acatcataga agttccagac 120gaaaaagcac cagaaaatat gagtgacaaa gaaattgagc aagtaaaaga aaaagaaggc 180caacgaatac tagccaaaat caaaccacaa tccacagtca ttacattaga aatacaagga 240aagatgctat cttccgaagg attggcccaa gaattgaacc aacgcatgac ccaagggcaa 300agcgactttg ttttcgtcat tggcggatca aacggcctgc acaaggacgt cttacaacgc 360agtaactacg cactatcatt cagcaaaatg acattcccac atcaaatgat gcgggttgtg 420ttaattgaac aagtgtacag agcatttaag attatgcgag gagaagcgta tcacaaataa 480aactaaaaaa tagattgtgt ataatataaa aggagcggat ttatattaaa actttgaatt 540caaaaattat tgaaagggaa gctaccttag aaattgaatc tatggcaact aatac 5958929DNAArtificial SequenceStaphylococcus aureus 89gtcaaaaatc atgaacctca ttacttatg 299028DNAArtificial SequenceStaphylococcus aureus 90ctatgtcaaa aatcatgaac ctcattac 289123DNAArtificial SequenceStaphylococcus aureus 91ggaggctaac tatgtcaaaa atc 239228DNAArtificial SequenceStaphylococcus aureus 92tcatgaacct cattacttat gataagnt 289321DNAArtificial SequenceStaphylococcus aureus 93gatagactaa ttatcttcat c 219421DNAArtificial SequenceStaphylococcus aureus 94cagactgtgg acaaactgat t 219520DNAArtificial SequenceStaphylococcus aureus 95tgagatcatc tacatcttta 209620DNAArtificial SequenceStaphylococcus aureus 96ggatcaaaag ctactaaatc 209720DNAArtificial SequenceStaphylococcus aureus 97atgctctttg ttttgcagca 209823DNAArtificial SequenceStaphylococcus aureus 98atgaaagact gcggaggcta act 239919DNAArtificial SequenceStaphylococcus aureus 99ggatcaaacg gcctgcaca 1910019DNAArtificial SequenceStaphylococcus aureus 100tcattggcgg atcaaacgg 1910122DNAArtificial SequenceStaphylococcus aureus 101acaacgcagt aactacgcac ta 2210222DNAArtificial SequenceStaphylococcus aureus 102taactacgca ctatcattca gc 2210322DNAArtificial SequenceStaphylococcus aureus 103acatcaaatg atgcgggttg tg 2210422DNAArtificial SequenceStaphylococcus aureus 104tcaaatgatg cgggttgtgt ta 2210524DNAArtificial SequenceStaphylococcus aureus 105caaatgatgc gggttgtgtt aatt 2410621DNAArtificial SequenceStaphylococcus aureus 106atcaaatgat gcgggttgtg t 2110720DNAArtificial SequenceStaphylococcus aureus 107accaaacgac atgaaaatca 2010818DNAArtificial SequenceStaphylococcus aureus 108gtttgatccg ccaatgac 1810923DNAArtificial SequenceStaphylococcus aureus 109gaggaccaaa cgacatgaaa atc 2311023DNAArtificial SequenceStaphylococcus aureus 110gccaaaatta aaccacaatc cac 2311124DNAArtificial SequenceStaphylococcus aureus 111cattttgctg aatgatagtg cgta 2411221DNAArtificial SequenceStaphylococcus aureus 112aagaattgaa ccaacgcatg a 2111321DNAArtificial SequenceStaphylococcus aureus 113aaacgacatg aaaatcacca t 2111421DNAArtificial SequenceStaphylococcus aureus 114tattgcaggt ttcgatgttg a 2111522DNAArtificial SequenceStaphylococcus aureus 115tgacccatat cgcctaaaat ac 2211622DNAArtificial SequenceStaphylococcus aureus 116aaaggacaac aaggtagcaa ag 2211722DNAArtificial SequenceStaphylococcus aureus 117tctgtggata aacaccttga tg 2211823DNAArtificial SequenceStaphylococcus aureus 118ggcataaatg tcaggaaaat atc 2311922DNAArtificial SequenceStaphylococcus aureus 119gtgggaaatg gctgttgttg ag 2212022DNAArtificial SequenceStaphylococcus aureus 120ttcgttccct ccattaactg tc 2212121DNAArtificial SequenceStaphylococcus aureus 121gttcaagccc agaagcgatg t 2112223DNAArtificial SequenceStaphylococcus aureus 122tcgggcataa atgtcaggaa aat 2312333DNAArtificial SequenceStaphylococcus aureus 123ttattaggta aaccagcagt aagtgaacaa cca 3312436DNAArtificial SequenceStaphylococcus aureus 124cgcttgccac atcaaatgat gcgggttgtg caagcg 3612536DNAArtificial SequenceStaphylococcus aureus 125cccaccccac atcaaatgat gcgggttgtg ggtggg 3612637DNAArtificial SequenceStaphylococcus aureus 126cccgcgcgta gttactgcgt tgtaagacgt ccgcggg 3712748DNAArtificial SequenceStaphylococcus aureus 127cgaccggatt cccacatcaa atgatgcggg ttgtgttaat tccggtcg 4812837DNAArtificial SequenceStaphylococcus aureus 128cccgcgcrta gttactrcgt tgtaagacgt ccgcggg 3712929DNAArtificial SequenceStaphylococcus aureus 129ccccgtagtt actgcgttgt aagacgggg 2913037DNAArtificial SequenceStaphylococcus aureus 130cccgcgcata gttactgcgt tgtaagacgt ccgcggg 3713137DNAArtificial SequenceStaphylococcus aureus 131cccgcgcgta gttactacgt tgtaagacgt ccgcggg 3713227DNAArtificial SequenceStaphylococcus aureus 132gtttttatca ccatattgaa tttatac 2713325DNAArtificial SequenceStaphylococcus aureus 133atttacttga aagactgcgg aggag 2513424DNAArtificial SequenceStaphylococcus aureus 134tgtttgagct tccacagcta tttc 2413527DNAArtificial SequenceStaphylococcus aureus 135ccctataatt ccaattattg cactaac 2713625DNAArtificial SequenceStaphylococcus aureus 136atgaggagat aataatttgg agggt 2513727DNAArtificial SequenceStaphylococcus aureus 137gctgaaaaaa ccgcatcatt trtgrta 2713827DNAArtificial SequenceStaphylococcus aureus 138gctgaaaaaa ccgcatcatt tatgata 2713928DNAArtificial SequenceStaphylococcus aureus 139gaaaaaaccg catcatttat gatatgnt 2814022DNAArtificial SequenceStaphylococcus aureus 140tatattgtgg catgatttct tc 2214123DNAArtificial SequenceStaphylococcus aureus 141cgaatggact agcactttct aaa 2314222DNAArtificial SequenceStaphylococcus aureus 142agacaacttt atgcaggtcc tt 2214322DNAArtificial SequenceStaphylococcus aureus 143taactgcttg ggtaacctta tc 2214429DNAArtificial SequenceStaphylococcus aureus 144atttcatata tgtaattcct ccacatctc 2914529DNAArtificial SequenceStaphylococcus aureus 145tttagtttta tttatgatac gcttctcca 2914625DNAArtificial SequenceStaphylococcus aureus 146ctctataaac atcgtatgat attgc 2514730DNAArtificial SequenceStaphylococcus aureus 147cctaattttt agttttattt atgatacgnt 3014835DNAArtificial SequenceStaphylococcus aureus 148cacaacctaa tttttagttt tatttatgat acgnt 3514923DNAArtificial SequenceStaphylococcus aureus 149atattctaga tcatcaatag ttg 2315029DNAArtificial SequenceStaphylococcus aureus 150caaatattat

ctcgtaattt accttgttc 2915126DNAArtificial SequenceStaphylococcus aureus 151ctactatgaa ctgtgcaatt tgttct 2615225DNAArtificial SequenceStaphylococcus aureus 152caatcggtat ctgtaaatat caaat 2515322DNAArtificial SequenceStaphylococcus aureus 153ttggttccat ctgaactttg ag 2215426DNAArtificial SequenceStaphylococcus aureus 154cacagaaatg taattttgga atgagg 2615529DNAArtificial SequenceStaphylococcus aureus 155ctctgcttta tattataaaa ttacggctg 2915624DNAArtificial SequenceStaphylococcus aureus 156tcgcatacct gtttatcttc tact 2415724DNAArtificial SequenceStaphylococcus aureus 157tttaaattca gctatatggg gaga 2415822DNAArtificial SequenceStaphylococcus aureus 158ttccgttttg ctattccata at 2215920DNAArtificial SequenceStaphylococcus aureus 159aaaagaaaga cggtgaaggc 2016025DNAArtificial SequenceStaphylococcus aureus 160cacttcatta tactgttttc tttgc 2516122DNAArtificial SequenceStaphylococcus aureus 161tcaccgtctt tcttttgacc tt 2216227DNAArtificial SequenceStaphylococcus aureus 162cactttttat tcttcaaaga tttgagc 2716328DNAArtificial SequenceStaphylococcus aureus 163atggaaattc ttaatcttta cttgtacc 2816424DNAArtificial SequenceStaphylococcus aureus 164agcatcttct ttacatcgct tact 2416521DNAArtificial SequenceStaphylococcus aureus 165ttgaggatca aaagttgttg c 2116621DNAArtificial SequenceStaphylococcus aureus 166cgatgatttt atagtaggag a 2116728DNAArtificial SequenceStaphylococcus aureus 167ttcaatctct aaatctaaat cagttttg 2816824DNAArtificial SequenceStaphylococcus aureus 168aggcgagaaa atggaacata tcaa 2416923DNAArtificial SequenceStaphylococcus aureus 169cagcaattcw cataaacctc ata 2317027DNAArtificial SequenceStaphylococcus aureus 170acaaactttg aggggatttt tagtaaa 2717124DNAArtificial SequenceStaphylococcus aureus 171ggatgtgggt atgctaatgt tgtt 2417227DNAArtificial SequenceStaphylococcus aureus 172tgaacaattt tatttctcat accatag 2717326DNAArtificial SequenceStaphylococcus aureus 173tcccctaatg atagctggta tatatt 2617427DNAArtificial SequenceStaphylococcus aureus 174tctagggaat caaagaaaag taatagt 2717526DNAArtificial SequenceStaphylococcus aureus 175ggtacaagta aagattaaga atttcc 2617625DNAArtificial SequenceStaphylococcus aureus 176actagaatct ccaaatgaat ccagt 2517724DNAArtificial SequenceStaphylococcus aureus 177tgataagcca ttcattcacc ctaa 2417824DNAArtificial SequenceStaphylococcus aureus 178aatggcttat caaagtgaat atgc 2417924DNAArtificial SequenceStaphylococcus aureus 179taatttcctt tttttccatt cctc 2418025DNAArtificial SequenceStaphylococcus aureus 180tgagatctgc tggaacaaaa gtgaa 2518120DNAArtificial SequenceStaphylococcus aureus 181cggtcgagtt tgctgaagaa 2018226DNAArtificial SequenceStaphylococcus aureus 182ctattgttcc acaaattatg attact 2618326DNAArtificial SequenceStaphylococcus aureus 183cttcttttct tgttattctt tcttct 2618423DNAArtificial SequenceStaphylococcus aureus 184gttgccatag attcaatttc tag 2318519DNAArtificial SequenceStaphylococcus aureus 185caccctgcaa gatatgttt 1918625DNAArtificial SequenceStaphylococcus aureus 186aatggaattt gttaatttca taaat 2518723DNAArtificial SequenceStaphylococcus aureus 187ttccgaagct aattctgtta ata 2318825DNAArtificial SequenceStaphylococcus aureus 188ttccgaagtc ataatcaatc aaatt 2518924DNAArtificial SequenceStaphylococcus aureus 189caaattgtag cattcttgaa ctat 2419023DNAArtificial SequenceStaphylococcus aureus 190gccaaaatca aaccacaatc aac 2319123DNAArtificial SequenceStaphylococcus aureus 191gccaaaatca aaccacaatc cac 231922150DNAArtificial SequenceStaphylococcus aureus 192tgaatcgttt aacgtgtcac atgatgcgat agatccgcaa ttttatattt tccataataa 60ctataagaag tttacgattt taacagatac gggttacgtg tctgatcgta tgaaaggtat 120gatacgtggc agcgatgcat ttatttttga gagtaatcat gacgtcgata tgttgagaat 180gtgtcgttat ccatggaaga cgaaacaacg cattttaggc gatatgggtc atgtatctaa 240tgaggatgcg ggtcatgcga tgacagacgt gattacaggt aacacgaaac gtatttactt 300atcgcattta tcacaagata ataatatgaa agatttggcg cgtatgagtg ttggccaagt 360attgaacgaa cacgatattg atacggaaaa agaagtattg ctatgtgata cggataaagc 420tattccaaca ccaatatata caatataaat gagagtcatc cgataaagtt ccgcactgct 480gtgaaacgac tttatcgggt gcttttttat gttgttggtg ggaaatggct gttgttgagt 540tgaatcggat tgattgaaat gtgtaaaata attcgatatt aaatgtaatt tataaataat 600ttacataaaa tcaaacattt taatataagg attatgataa tatattggtg tatgacagtt 660aatggaggga acgaaatgaa agctttatta cttaaaacaa gtgtatggct cgttttgctt 720tttagtgtga tgggattatg gcatgtctcg aacgcggctg agcagcatac accaatgaaa 780gcacaagcag caacaacaga taagcaacaa gtaacgccaa caaaggaagc ggctcatcaa 840tctggtgaag aagcggcaac caacgtatca gcatcagtac agggaacagc tgatgataca 900aacaacaaag taacatccaa cgcaccatct aacaaaccat ctacagcagt ttcaacaaca 960gtaaacgaaa cgcgcgacgt agatgcacaa caagcctcaa cacaaaaacc aactcaatca 1020gcaacattca aattatcaaa tgctaaaaca gcatcacttt caccacgaat gtttgctgct 1080aatgcaccac aaacaacaac acataaaata tggttctgtt gcaaagtaaa aaaatatagc 1140taaccactaa tttatcatgt cagtgttcgc ttaacttgct agcatgatgc taatttcgtg 1200gcatggcgaa aatccgtaga tctgaagaga cctgcggttc tttttatata gagcgtaaat 1260acattcaata ccttttaaag tattctttgc tgtattgata ctttgatacc ttgtctttct 1320tactttaata tgacggtgat cttgctcaat gaggttattc agatatttcg atgtacaatg 1380acagtcaggt ttaagtttaa aagctttaat tactttagcc attgctacct tcgttgaagg 1440tgcctgatct gtaattacct tttgaggttt accaaattgt ttaatgagac gtttgataaa 1500cgcatatgct gaatgattat ctcgttgctt acgcaaccaa atatctaatg tatgtccctc 1560tgcatcaatg gcacgatata aatagctcca ttttcctttt attttgatgt acgtctcatc 1620aatacgccat ttgtaataag cttttttatg ctttttcttc caaatttgat acaaaattgg 1680ggcatattct tgaacccaac ggtagaccgt tgaatgatga acgtttacac cacgttccct 1740taatatttca gatatatcac gataactcaa tgtatatctt agatagtagc caacggctac 1800agtgataaca tccttgttaa attgtttata tctgaaatag ttcatacaga agactccttt 1860ttgttaaaat tatactataa attcaacttt gcaacagaac cgaaaaacta gacttgatta 1920caaaatggag cttgggacat aaatgatttt ttaaaaatga gatgagacgt agattaactc 1980cataatcaat acgaatctat cgacttcttt atttatgata ttcatctctt tttaatggaa 2040ataaaagtgc gattaatgtg ataatacagt tacgttaatt aaaaaaataa aaatgcaagg 2100agaggtaata tgctaactgt atatggacat agaggattac ctagtaaagc 215019323DNAArtificial SequenceStaphylococcus aureus 193gccaaaatta aaccacaatc cac 2319424DNAArtificial SequenceStaphylococcus aureus 194ctcccatttc ttccaaaaaa tata 2419529DNAArtificial SequenceStaphylococcus aureus 195caaatattat ctcgtaattt accttgttc 2919629DNAArtificial SequenceStaphylococcus aureus 196ctctgcttta tattataaaa ttacggctg 2919722DNAArtificial SequenceStaphylococcus aureus 197caagctccat tttgtaatca ag 2219822DNAArtificial SequenceStaphylococcus aureus 198caagctccat tttgtaatca ag 2219926DNAArtificial SequenceStaphylococcus aureus 199atgtcaaaaa tcatgaacct cattac 26200686DNAArtificial SequenceStaphylococcus aureus 200atgacagaat acttattaag tgctggcata tgtatggcaa ttgtttcaat attacttata 60gggatggcta tcagtaatgt ttcgaaaggg caatacgcaa agaggttttt ctttttcgct 120actagttgct tagtgttaac tttagttgta gtttcaagtc taagtagctc agcaaatgca 180tcacaaacag ataacggcgt aaatagaagt ggttctgaag atccaacagt atatagtgca 240acttcaacta aaaaattaca taaagaacct gcgacattaa ttaaagcgat tgatggtgat 300acggttaaat taatgtacaa aggtcaacca atgacattca gactattatt agttgataca 360cctgaaacaa agcatcctaa aaaaggtgta gagaaatatg gccctgaagc aagtgcattt 420acgaaaaaaa tggtagaaaa tgcaaataaa attgaagtcg agtttgacaa aggtcaaaga 480actgataaat atggacgtgg cttagcgtat atttatgctg atggaaaaat ggtaaacgaa 540gctttagttc gtcaaggctt ggctaaagtt gcttatgttt ataaacctaa caatacacat 600gaacaacttt taagaaaaag tgaagcacaa gcgaaaaaag agaaattaaa tatttggagc 660gaagacaacg ctgattcagg tcaata 68620129DNAArtificial SequenceStaphylococcus aureus 201atttcatata tgtaattcct ccacatctc 2920237DNAArtificial SequenceStaphylococcus aureus 202cccgcgcgta gttactgcgt tgtaagacgt ccgcggg 3720337DNAArtificial SequenceStaphylococcus aureus 203cccgcgcata gttactgcgt tgtaagacgt ccgcggg 3720437DNAArtificial SequenceStaphylococcus aureus 204cccgcgcgta gttactacgt tgtaagacgt ccgcggg 3720518DNAArtificial SequenceStaphylococcus aureus 205ggatcaaacg gcctgcac 18

Claims

1. A method of specifically detecting the presence of a Staphylococcus aureus (S. aureus) strain and identifying a methicillin-resistant S. aureus strain from a clinical sample in a single assay, comprising contacting said sample with at least one primer and/or probe of at least 11 nucleotides that anneals under stringent conditions to an S. aureus-specific sequence within SEQ ID NO: 200 or the complement thereof, or any sequence which differs from SEQ ID NO: 200 by 1 to 20 nucleotides; contacting said sample with at least one primer and/or probe of at least 10 nucleotides that anneals under stringent conditions to at least one of the following MREJ specific SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88 or the complement thereof; and detecting the presence and/or amount of annealed probe(s), or detecting the amount of an amplification product produced through annealing of the primers to the nucleic acids, as an indication of the presence and/or amount of S. aureus, and as an indication of the presence and/or amount of MRSA, wherein said stringent conditions comprise 4 mM MgCl₂, 100 mM Tris (pH 8.3), 10 mM KCl, 5 mM (NH₄)₂SO₄, 0.15 mg/mL BSA, 4% trehalose at 59.degree. C.

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