# Patent

## Title
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.

## Background
RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Application Ser. No. 60/870,823, filed on Dec. 19, 2006, by Jean et al. entitled “DETECTION OF STAPHYLOCOCCUS AUREUS AND IDENTIFICATION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS,” which is hereby expressly incorporated by reference in its entirety.


SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled GENOM.072A.TXT, created Nov. 29, 2007, which is 124 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
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. 
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.
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.
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.
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 chromosomal 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).
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).
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 EVIGENETM MRSA Detection kit.
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.
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 Ito 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.
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).
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.
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).
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.
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.
Poulsen et al. describe detection of methicillin resistance in coagulase-negative Staphylococci and in S. aureus using the EVIGENETM 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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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:
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; and
SEQ ID NOs: 4 and 8, or the complements thereof.
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:
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; and
SEQ ID NOs: 4 and 8.
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:
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
SEQ ID NOs: 99 and 163, or the complement thereof.
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:
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
SEQ ID NOs: 99 and 163.
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
SEQ ID NOs: 1 and 6
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
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:
SEQ ID NOs: 1 and 6
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
SEQ ID NOs: 99 and 163.
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
SEQ ID NOs: 3 and 8
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
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:
SEQ ID NOs: 3 and 8
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
SEQ ID NOs: 99 and 163.
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.
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.
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.
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.
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.
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
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.
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
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.
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
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.
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
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 normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as NEUGENETM 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.
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.
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-.beta.-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-.beta.-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-.beta.-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.
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
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.
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 (Tm) than do shorter ones, and are less likely to be repeated within a given target sequence, thereby minimizing promiscuous hybridization.
As used herein, “Tm” 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 Tm of polynucleotides are well known in the art. For example, the Tm may be calculated by the following equation: Tm=69.3+0.41 x.(G+C)%−6−50/L, wherein L is the length of the probe in nucleotides. The Tm of a hybrid polynucleotide may also be estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used for calculating Tm 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 Tm. A calculated Tm is merely an estimate; the optimum temperature is commonly determined empirically.
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%.
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.
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.
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.
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.
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
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.
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.
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.).
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.
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.
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).
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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).
Accordingly, some embodiments comprise, consist essentially of, or consist of primer pairs that hybridize under stringent conditions to the nucleic acid sequences of:
SEQ ID NOs: 1 and 6
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
SEQ ID NOs: 99 and 163, or the complements thereof.
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:
SEQ ID NOs: 3 and 8;
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
SEQ ID NOs: 99 and 163, or the complements thereof.
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.
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).
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:
SEQ ID NOs: 1 and 6
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
SEQ ID NOs: 99 and 163, or the complements thereof.
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:
SEQ ID NOs: 3 and 8;
SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 144;
SEQ ID NOs: 99 and 150;
SEQ ID NOs: 99 and 155; and
SEQ ID NOs: 99 and 163, or the complements thereof.
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.
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
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 FASTSTARTTM Taq DNA polymerase (Roche), 50 mM Tris (EMD), 10 mM KCl (Laboratoire Mat), and 5 mM (NH4)2SO4 (SIGMA).
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).
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).
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:
SEQ ID NOs: 1 and 5
SEQ ID NOs: 1 and 6
SEQ ID NOs: 2 and 6
SEQ ID NOs: 4 and 7
SEQ ID NOs: 4 and 8
SEQ ID NOs: 3 and 7, and
SEQ ID NOs: 3 and 8.
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
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.
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).
PRC 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.
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.
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.
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
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.
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.
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 2









PCR results















S. aureus



Strain
MRSA
IC
(Texas


Species
number
(FAM)
(TET)
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
ATCC 19433
−
+
−


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
ATCC 10953
−
+
−


subsp. 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
ATCC 27337
−
+
−


anaerobius






Peptostreptococcus
LSPQ 2639
−
+
−


asaccharolyticus






Porphyromonas
ATCC 25260
−
+
−


asaccharolytica






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
R570
−
+
−


subsp. Urealyticum






Staphylococcus delphini
ATCC 49171
−
+
−


Staphylococcus epidermidis
ATCC 35984
−
+
−


Staphylococcus equorum
ATCC 43958
−
+
−


Staphylococcus fells
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 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









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
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.
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.
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 VITEKTM bacterial identification system (bioMérieux, Durham, N.C.).
DNA was isolated from the isolates using the IDITM 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.
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.
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:
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.
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).
The data collected are depicted in Tables 4A, 4B and 4C, below.







TABLE 4A







(A)









PCR

















Unre-



MRSA
MSSA
Negative
Total
solved

















Cul-
MRSA
 27 (32)*
0 (0)
0 (0)
27 (32)
1 (0)


ture
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 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 4C


























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.
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.
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.
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.
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.
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.

## 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 sample in a single assay, comprising:
simultaneously contacting said sample with
at least one primer pair that is capable of amplifying a sequence from a nuc gene S. aureus when an S. aureus strain is present in the sample to produce a nuc amplicon, said primer pair comprising a first and a second nuc primer, said first and second nuc primers being between 11 and 45 nucleotides in length and being capable of annealing under PCR conditions to opposite strands of S. aureus chromosomal DNA within the nuc gene; and
at least one MREJ primer pair comprising a first and second MREJ primer that is capable of amplifying a polymorphic mec right extremity junction (MREJ) sequence of a methicillin resistant S. aureus (MRSA) strain when an MRSA strain is present in the sample to produce an MREJ amplicon, said MREJ sequence comprising polymorphic sequences from the right extremity of an SCCmec cassette inserted into the S. aureus chromosome and S. aureus chromosomal DNA, said first and second MREJ primers being between 11 and 45 nucleotides in length and being capable of annealing under PCR conditions to opposite strands of MRSA chromosomal DNA, to create an amplification mixture,
wherein said PCR 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° C. subjecting the amplification mixture to an amplification protocol to enable amplification of both nuc and MREJ sequences, thereby producing an amplified sample; and

detecting the presence and/or amount of nuc amplicons and MREJ amplicons in the amplified sample as an indication of the presence and or amount of S. aureus and MRSA, respectively, present in the sample.

2. The method of claim 1, wherein said nuc gene comprises SEQ ID NO: 200.

3. The method of claim 1, wherein said assay further comprises
providing an internal control nucleic acid sequence to said sample, wherein said nuc primer pair and/or said MREJ primer pair produce an internal control amplicon under said amplification protocol.

4. The method of claim 1, wherein said MREJ sequence is selected from the group consisting of an MREJ type i, type ii, type iii, type iv, type v, type vi, type vii, type viii, type ix, and type x sequence, or any combination thereof.

5. The method of claim 4, wherein said MREJ sequence is a type i sequence, wherein SEQ ID NO:14 comprises said MREJ type i sequence.

6. The method of claim 4, wherein said MREJ sequence is a type ii sequence, wherein SEQ ID NO: 23 comprises said MREJ type ii sequence.

7. The method of claim 4, wherein said MREJ sequence is a type iii sequence, wherein SEQ ID NO:43 comprises said MREJ type iii sequence.

8. The method of claim 4, wherein said MREJ sequence is a type iv sequence, wherein SEQ ID NO: 59 comprises said MREJ type iv sequence.

9. The method of claim 4, wherein said MREJ sequence is a type v sequence, wherein SEQ ID NO:65 comprises said MREJ type v sequence.

10. The method of claim 4, wherein said MREJ sequence is a type vi sequence, wherein SEQ ID NO: 69 comprises said MREJ type vi sequence.

11. The method of claim 4 wherein said MREJ sequence is a type vii sequence, wherein SEQ ID NO:70 comprises said MREJ type vii sequence.

12. The method of claim 1, wherein said detecting the presence and/or amount of nuc amplicons in the amplified sample comprises contacting the amplified sample with a nuc probe that anneals under said PCR conditions to said nuc amplicon, wherein said nuc probe comprises a detectable moiety.

13. The method of claim 1, wherein said detecting the presence and/or amount of MREJ amplicons in the amplified sample comprises contacting the amplified sample with an MREJ probe that anneals under said PCR conditions to said MREJ amplicon, wherein said MREJ probe comprises a detectable moiety.

14. The method of claim 4, wherein said MREJ sequence comprises type i, ii, iii, iv, v and vii MREJ sequences.

15. The method of claim 14, wherein said at least one MREJ primer pairs anneal under said PCR conditions to the nucleic acid sequences selected from the group consisting of:
SEQ ID NOs: 99 and 199 or the full complements of SEQ ID NOs: 99 and 199;
SEQ ID NOs: 99 and 201 or the full complements of SEQ ID NOs: 99 and 201;
SEQ ID NOs: 99 and 195 or the full complements of SEQ ID NOs: 99 and 195;
SEQ ID NOs: 99 and 155 or the full complements of SEQ ID NOs: 99 and 155; and
SEQ ID NOs: 99 and 163 or the full complements of SEQ ID NOs: 99 and 163.

16. The method of claim 1, wherein said first and second nuc primers comprise oligonucleotides that anneal under said PCR conditions to at least 11consecutive nucleotides of sequences selected from the group consisting of:
SEQ ID NOs: 1 and 6, or the full complements of SEQ ID NOs: 1 and 6;
SEQ ID NOs: 2 and 5, or the full complements of SEQ ID NOs: 2 and 5;
SEQ ID NOs: 2 and 6, or the full complements of SEQ ID NOs: 2 and 6;
SEQ ID NOs: 3 and 7, or the full complements of SEQ ID NOs: 3 and 7;
SEQ ID NOs: 4 and 7, or the full complements of SEQ ID NOs: 4 and 7; and
SEQ ID NOs: 4 and 8, or the full complements of SEQ ID NOs: 4 and 8.

17. The method of claim 16, wherein said nuc primer pair comprises a first primer that anneals under said PCR conditions to SEQ ID NO: 1, or the complement of SEQ ID NO: 1 and a second primer that anneals under said PCR conditions to SEQ ID NO: 6 or the complement of SEQ ID NO: 6.

18. The method of claim 17, further comprising providing a probe that anneals under said PCR conditions to a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, the full complement of SEQ ID NO: 9, and the full complement of SEQ ID NO: 10.

19. The method of claim 16, wherein said nuc primer pair comprises a first primer that anneals under said PCR conditions to SEQ ID NO: 3 or the full complement of SEQ ID NO: 3, and a second primer that anneals under said PCR conditions to SEQ ID NO: 8 or the full complement of SEQ ID NO: 8.

20. The method of claim 19, further comprising providing a probe that anneals under said PCR conditions to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, the full complement of SEQ ID NO.: 11, and the full complement of SEQ ID NO: 12.

21. The method of claim 13, wherein said MREJ probe comprises an oligonucleotide that anneals under said PCR conditions to at least 11 consecutive nucleotides of a sequence selected from the group consisting of: SEQ ID NO:126, 128, 130, 131, 204, the full complement of SEQ ID NO: 126, the full complement of SEQ ID NO: 128, the full complement of SEQ ID NO: 130, the full complement of SEQ ID NO: 131, and the full complement of SEQ ID NO: 204.