WO2008061376A1

WO2008061376A1 - Methods and kits for detecting methicillin-resistant staphylococcus aureus

Info

Publication number: WO2008061376A1
Application number: PCT/CA2007/002122
Authority: WO
Inventors: Daniel Gaudet; François PARADIS; Doria Grimard
Original assignee: Boreal Pharma Recherche Clinique Inc
Current assignee: Boreal Pharma Recherche Clinique Inc
Priority date: 2006-11-23
Filing date: 2007-11-23
Publication date: 2008-05-29
Anticipated expiration: 2009-05-23
Also published as: CA2569552A1; WO2008061376B1

Abstract

The invention relates to the identification of microbial nucleotide sequences for the determination of the presence or absence of a particular microorganism. More particularly, the present invention is concerned with diagnostic methods and kits which can promptly detect and identify in a sample or specimen antibiotic-resistant microorganisms such as methicillin-resistant Staphylococcus aureus (MRSA).

Description

Methods and kits for detecting methicillm-resistant Staphylococcus aureus

Related Application [0001] This application claims priority to Canadian patent application no. CA 2,569,552 filed November 23, 2006, which is incorporated herein by reference.

Field of the Invention

[0002] The present invention relates to the field of microbiology and particularly to the identification of microbial nucleotide sequences as a diagnostic method for the determination of the presence or absence of a particular microorganism. More particularly, the present invention is concerned with diagnostic methods and kits which can promptly detect and identify methicillin-resistant Staphylococcus aureus (MRSA).

Background of the Invention

[0003] It is not uncommon to observe development of microbial resistance soon after the introduction of a new antibiotic. Methicillin-resistant Staphylococcus aureus (MRSA) strains were soon identified after the introduction of methicillin, which itself was developed to overcome resistance to penicillin. MRSA emerged in the 1980s as a major clinical and epidemiologic problem in hospitals. MRSA are resistant to all ^-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. Consequently, there is a need to develop rapid and simple screening or diagnostic tests for detection and/or identification of MRSA to reduce its dissemination and improve the diagnosis and treatment of infected patients.

[0004] 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 supernumerary /Mactam-resistant penicillin-binding protein (PBP), which takes over the biosynthetic functions of the normal PBPs when the cell is exposed to /Mactam 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 PBP2¹), is encoded by the chromosomal mecA gene and functions as a /Mactam- 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. The mecA gene is carried by a genetic element, designated staphylococcal cassette chromosome mec (SCCmec), which is inserted into a specific S. aureus chromosomal site in the same orientation.

[0005] Methods to detect and identify MRSA based on the detection of the mecA gene and S. aureus-specific chromosomal sequences have been described. For instance, U.S. patent No. 6,156,507 (Hiramatsu et al.), published patent applications US2006/0252078 and US 2007/0082340 describe PCR- based assays for the amplification of chromosomal regions specific to MRSA. Those assays utilize primers that hybridize to the right extremities of the 3 types of SCCmec DNAs 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. However, to achieve a rapid detection of MRSA, the amplification of the desired chromosomal regions is carried out in real time, an approach which requires an expensive PCR apparatus and expensive reactants.

[0006] There are also numerous methodologies that allow multiplexing for the simultaneous detection of multiple nucleic acid sequences in a single reaction vessel. One of these is the microsphere-based suspension array technologies, such as the Luminex® xMAP™ system. This technology is being utilized with increasing frequency for nucleic acid detection in the areas of single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, HLA DNA typing and microbial detection (Dunbar S., Clinica Chimica Acta 363 (2006) 71-82). Before the present invention, the Luminex® technology had never been applied to the identification of antibiotic-resistant microorganism by detecting two different sets of bacterial chromosomal regions, one of these regions including nucleic acid overlapping both sides of the SCCmec integration site.

[0007] In view of the above, there is thus a need for methods and kits for a quick, sensitive, specific, reliable and cheap detection of antibiotic-resistant microorganisms, and more particularly detection and identification of methicillin- resistant Staphylococcus aureus (MRSA).

Summary of the Invention

[0008] The present invention provides methods and kits for detecting antibiotic-resistant microorganisms.

[0009] Accordingly, an aspect of the present invention relates to methods and kits to identify the presence of methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample, e.g. a nose swab from a patient. As is known, MRSA strains have a genome with a SCCmec inserted at an integration site in their genome.

[0010] In one embodiment, the method comprises:

- amplifying a first region of the genome of the MRSA strain, the first region comprising and extending on both sides of the integration site;

- amplifying a second region of the genome of the MRSA strain, the second region being specific to S. aureus and being also distinct from the first region;

- identifying the presence of the MRSA strain in the sample by detecting the presence of both amplicons.

[0011] In another embodiment, the method comprises -amplifying a first region of the genome of the MRSA strain, the first region comprising the integration site and extending from a first position in the SCCmec to a second position in said genome;

-amplifying a second region of the genome of said strain, said second region being specific to S. aureus and extending from third and forth positions in said genome, wherein the second, third and a forth positions are selected for priming under suitable amplification conditions the production of two distinct amplicons corresponding to said first and second region;

-identifying the presence of said MRSA strain in said sample by detecting the presence of both amplicons.

[0012] In yet another embodiment, the method comprises using a set of primers for amplifying first and second regions of the MRSA genome, the first region comprising the integration site and extending from a first position in the SCCmec to a second position in the MRSA genome; the second region being specific to S. aureus and extending from third and forth positions in the MRSA genome, wherein the primers are selected for priming under suitable amplification conditions the production of two distinct amplicons of about 50 bp to about 20 kb corresponding to the first and second region, and; hybridizing to the amplicons first and second labelled probes specific for the first and second region, respectively; detecting the labelled probes hybridized to a corresponding amplicon, wherein detection of each of the hybridized labelled probes comprises using at least two different quantities thereof, measuring for each one of these at least two quantities a detection signal, and comparing these detection signals, wherein a reliable difference in the detection signals is indicative of the presence of the amplicon to be detected; whereby it is the presence of both of said amplicons which is indicative of the presence of the MRSA strain in the sample.

[0013] Preferably, the first, second, third and a forth positions are selected such that said the amplicons products of the amplification comprises about 50 bp to about 20 kb. Preferably also, the second, third and a forth positions are also selected such that the second region does not overlap the first region, thereby avoiding an undesirable hybridation between any complementary region of the amplicons generated. As used herein, the term "two distinct amplicons" refers to two amplicons having totally different or at least substantially different sequences, with minimal overlapping sequences, if any. Overlapping sequences may exist but those overlapping sequences should not be sufficient for the complementary strands to hybridize to each other under typical amplification conditions.

[0014] The present invention further relates to kits for identifying the presence of methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample. In one embodiment, the kit comprises primers for amplifying first and second regions of the genome of the MRSA strain, the first region comprising and extending on both sides of the integration site, the second region being specific to S. aureus and being also distinct from the first region, wherein these primers are selected for priming under suitable amplification conditions the production of two distinct amplicons of about 50 bp to about 20 kb corresponding to the first and second regions.

[0015] In preferred embodiments of the methods and kit according to the invention, the second position is located inside the orfX gene, the gene into which SCCmec is integrated. The second region to be amplified preferably comprises also a part (i.e. a section) of the orfX gene. However, it is preferable according to the invention that the first and second regions amplified do not overlap each other.

[0016] According to some embodiments, the preferred technique for the amplification of the first and second regions is polymerase chain reaction (PCR) technique. Under preferred conditions, the amplification of both regions is carried out in a single reaction vessel (i.e. multiplex reaction).

[0017] According to preferred embodiments, the detection of the each amplicon produced comprises using at least two different quantities of each amplicon for comparison purposes. By measuring a reliable difference in the detection signals obtained for each quantity, one can conclude that the amplicon to be detected (i.e. one of the two amplified bacterial chromosomal region) is present. As used herein, the term "reliable difference" refers to a true difference in the eye of those skilled in the art. A reliable difference is preferably significant and takes into account the limitation of the detection technique (e.g. background, sample variation, etc.).

[0018] As indicated hereinbefore, a definitive conclusion about the presence of the MRSA strain in the sample requires the amplification of both chromosomal regions which is eventually detected by the presence of both of amplicons. In preferred embodiments, the presence or absence of the two amplicons is detected using labelled probes which each hybridizes specifically to a corresponding amplicon. More preferably, each labelled probe comprises of a nucleic acid molecule that hybridizes specifically to the amplicon to be detected, said nucleic acid molecule being coupled to microspheres. The labelled probes thus form two distinct populations of microspheres. In preferred embodiments the two distinct populations of microspheres are easily distinguishable because the microspheres are dyed with two spectrally distinct fluorochromes for a quick and easy detection using the Luminex® microspheres technology.

[0019] Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention.

Brief Description of the Drawings

[0020] FIGURE 1 is a schematic representation of a PCR strategy according to one embodiment of the detection method according to the invention.

[0021] FIGURE 2 is bar graph showing median fluorescence intensity (MFI) of increasing amounts of orfX PCR product, according to experiments carried out for optimizing various conditions used in one embodiment of the detection method of the invention.

[0022] FIGURE 3 is bar graph showing median fluorescence intensity of increasing amounts of mecA/orfX junction PCR product, according to experiments carried out for optimizing various conditions used in one embodiment of the detection method of the invention.

[0023] FIGURE 4 is bar graph showing median fluorescence intensity of MRSA negative and MRSA positive sample and specimen according to an embodiment of the detection method of the invention.

Detailed Description of the Invention

[0024] Microorganisms resistant to antibiotics pose a serious health threat to individuals and the need for rapid and simple methods and kits for the detection and identification of those microorganisms is readily apparent. Although some tests already exist on the market, those tests typically require an expensive amplification apparatus. Each individual test is also costly because of the complex and expensive reactants which are required during the amplification step.

[0025] Accordingly, the present invention is concerned with diagnostic methods and kits which can promptly detect and identify antibiotic-resistant microorganisms and more particularly methicillin-resistant Staphylococcus aureus (MRSA) in a sample or specimen (e.g. nose swab, groin swab, bacterial colonies, liquid cultures, expectorations, skin wounds, throat swabs, blood samples, contaminated surfaces and liquids).

[0026] Resistance to methicillin is mediated by the presence of penicillin- binding protein 2a (PBP-2a) which is encoded by the mecA gene. Molecular studies and the completion of the whole genome sequences of three MRSA strains have shown that the mecA gene is located on a unique genomic island, the 21- 60-kb staphylococcal cassette chromosome mec (SCCmec). SCCmec is integrated at a fixed site on the chromosome (orfX) near the origin of replication. Its function is still unknown and it is present in S. aureus as well as in coagulase- negative straphylococci (CNS). So far, four different type of this mobile genetic element were found and designated type-l (NCTC 10442), type-ll (N315), type-Ill (85/2082) and type-IV with the subtypes Iva (JCSC 1968), IVb (JCSC 1978), and IVc (MRI 08).

[0027] In general terms, the present invention is based on the amplification and detection of at least two specific microbial genomic sequences. Those specific microbial genomic sequences are: 1 ) genomic sequences specific the bacteria to be detected (e.g. S. aureus) and 2) specific genomic sequences corresponding to a region of the gene(s) or transposon(s) responsible for the bacterial resistance to the antibiotic (e.g. the mecA gene responsible for the resistance to methicillin).

[0028] The disclosure of the present invention focuses primarily on the detection and identification of MRSA, and more particularly in the amplification and detection of a region overlapping the integration site of SCCmec which includes the mecA gene, the gene responsible for methicillin resistance, and the amplification and detection of a part of the S. aureus gene orfX. The mecA gene was chosen because it is absent from methicillin-sensitive S. aureus but is widely distributed among other species of staphylococci and is highly conserved. Interestingly, the mecA gene is always inserted in the same orientation into a specific S. aureus chromosomal site of the gene orfX (referred hereinafter as the "integration site"), making orfX the most convenient choice for the selection of a desired specific S. aureus genomic sequence. However, it is conceivable according to the general principles of the invention to use other S. aureus specific genomic sequences, for instance the nuc gene. Those skilled in the art will also understand that the principles of the present invention can be readily applied to the detection and identification of other antibiotic-resistant microorganisms wherein the bacterial resistance to the antibiotic is associated with genetic marker(s) or gene(s) which are inserted in specific chromosomal site(s) of the bacterial genome. Non-limitative examples includes Tn2009 carrying the tetM gene of Streptococcus pneumonia which is integrated in the Tn916-like genetic element (see Del Grosso M et al., Antimicrob. Agents Chemother 2004; 48:2037- 42). [0029] In one embodiment, the method of the invention comprises:

[0030] In another embodiment, the method comprises amplifying a first region of the genome of the MRSA strain, the first region comprising the integration site and extending from a first position in the SCCmec to a second position in said genome; amplifying a second region of the genome of said strain, said second region being specific to S. aureus and extending from third and forth positions in said genome, wherein the second, third and a forth positions are selected for priming under suitable amplification conditions the production of two distinct amplicons corresponding to said first and second region; identifying the presence of said MRSA strain in said sample by detecting the presence of both amplicons.

[0031] In yet another embodiment, the method comprises using a set of primers for amplifying first and second regions of the MRSA genome, the first region comprising the integration site and extending from a first position in the SCCmec to a second position in the MRSA genome; the second region being specific to S. aureus and extending from third and forth positions in the MRSA genome, wherein the primers are selected for priming under suitable amplification conditions the production of two distinct amplicons of about 50 bp to about 20 kb corresponding to the first and second region, and; hybridizing to the amplicons first and second labelled probes specific for the first and second region, respectively; detecting the labelled probes hybridized to a corresponding amplicon, wherein detection of each of the hybridized labelled probes comprises using at least two different quantities thereof, measuring for each one of these at least two quantities a detection signal, and comparing these detection signals, wherein a reliable difference in the detection signals is indicative of the presence of the amplicon to be detected; whereby it is the presence of both of said amplicons which is indicative of the presence of the MRSA strain in the sample.

[0032] According to some embodiments, the method of the invention comprises the uses specific primers in the method of polymerase chain reaction (PCR) for amplifying two selected specific regions of the genome of methicillin- resistant Staphylococcus aureus. Thereafter, the two regions so amplified are detected, preferably by using specific probes comprising nucleic acid-coupled microspheres suitable for detection using the Luminex® technology.

[0033] As used herein, the products of amplification of the regions amplified are referred as "amplicons". More specifically, the term "amplicon" refers to the product of the amplification reaction generated through the extension of either or both of a pair of amplification primers. An amplicon may contain exponentially amplified nucleic acids if both primers utilized hybridize to a target sequence. Alternatively, amplicons may be generated by linear amplification if one of the primers utilized does not hybridize to the target sequence. Thus, this term is used generically herein and does preferably, but not necessarily, implies the presence of exponentially amplified nucleic acids.

[0034] 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 non-nucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (such as Neugene™ polymers), and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.

[0035] The terms nucleotide and polynucleotide include, for example, 3'- deoxy^'.δ'-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., aminoalkylphosphoramidates, 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.

[0036] 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-/?-D-ribofuranosyl-purine) may be modified to form isoguanosine (2-oxy-6-amino-9-/?-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-/?-D-ribofuranosyl-2-oxy- 4-amino-pyrimidine) to form isocytosine (1-/?-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.

[0037] As exemplified hereinafter, specific binding or annealing of the primers and/or probes to nucleic acid sequences is accomplished through specific 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 the conditions used to detect the presence of the MRSA nucleic acids.

[0038] As well known in the art, 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) x 2°C +(number of G+C) x 4 ⁰C]. 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.

[0039] 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. The use of TMAC buffer during hybridization reduces significantly the effect of the G+C content of the hybridizing DNA molecules.

[0040] 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 5 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 ⁰C depending upon the lengths and/or the nucleic acid composition of the probes. 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. "Stringent hybridization conditions" refers to either or both of the following: a) 6x SSC at about 45°C, followed by one or more washes in 0.2x SSC, 0.1 % SDS at 65 ⁰C, and b) 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 ⁰C or 70°C for 12-16 hours, followed by washing.

[0041] 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.

[0042] In some embodiments, more than one primer and/or probe is provided. For example, some embodiments relate to methods for detecting a plurality of MRSA strains. A plurality of primers and/or probes may be used in reactions conducted in separate physical enclosures or in the same physical enclosure. Reactions testing for a variety of MRSA types can be conducted one at a time, or simultaneously. In embodiments where the plurality of primers is provided in the same physical enclosure, a multiplex PCR reaction can be conducted, with a plurality of oligonucleotides, most preferably that are all capable of annealing with a target region under common conditions. Figure 1 and the exemplification section hereinafter provides an example of such a multiplex PCR reaction using primers adapted for detecting several different types of SCCmec corresponding to types i to vi. However, it is conceivable according to the invention to detect more types of SCCmec by using additional primers selected or adapted for use in separate or multiplexed amplifications. [0043] It is also conceivable according to the invention to use in a multiplex PCR reaction a plurality of primers and/or probes that are specific for different strains of MRSA such that the exact type or strain of MRSA can be determined. The primers and/or probes used for detection can have different labels, to enable to distinguish one strain of MRSA from another MRSA strain. As used herein, the term "label" refers to entities capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin and the like.

[0044] Although the selection and evaluation of oligonucleotides suitable for diagnostic purposes requires much effort, it is quite possible for the individual skilled in the art to derive, from the selected DNA fragments, oligonucleotides other than the primers and probes described herein.

[0045] The present invention further encompass the use of additional specific and useful sequences for the primers and probes of the invention, including the sequences described in U.S. Patent No. 6,156,507, US application US 2004/0053213; US 2006/0252069; US 2006/0252078; US 2005/019893; EP 1 529 847; lto et al., 2001 , Antimicrob. Agents Chemother. 45:1323-1336; Huletsky et al., 2004, J Clin. Microbiol. 42:1875-1884; Ma et al, 2002, Antimicrob. Agents Chemother. 46:1147-1152; lto et al, Antimicrob Agents Chemother. 2004. 48:2637-2651 ; Oliveira et al, 2001 , Microb. Drug Resist. 7:349-360). Additional MRSA specific sequences other than those explicitly described herein and which are appropriate for detection and/or identification of MRSA may also be derived from the novel sequences yet to be discovered (e.g. new strains of MRSA) and are included within the scope of the invention.

[0046] Further, variants of the oligonucleotides disclosed herein can be designed. The oligonucleotide primers or probes may be shorter (most preferably of a length of at least 10 nucleotides) or longer than the ones chosen; they may also be selected somewhere else in the bacterial genome. If the target DNA or a variant thereof hybridizes to a given oligonucleotide, or if the target DNA or a variant thereof can be amplified by a given oligonucleotide PCR primer pair, the converse is also true; a given target DNA may hybridize to a variant oligonucleotide probe or be amplified by a variant oligonucleotide PCR primer. Alternatively, the oligonucleotides may be designed from the bacterial genome sequences for use in amplification methods other than PCR.

[0047] Oligonucleotides, including probes for hybridization and primers for DNA amplification, can be evaluated for their suitability for hybridization or PCR amplification by computer analysis using publicly and commercially available computer software, such as the Genetics Computer Group GCG Wisconsin package programs, and the Oligo™ 6 and MFOLD™ 3.0 primer analysis software. The potential suitability of the PCR primer pairs may also be evaluated prior to their synthesis by verifying the absence of unwanted features such as long stretches of one nucleotide and a high proportion of G or C residues at the 3¹ end (Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). Oligonucleotide amplification primers can be synthesized using an automated DNA synthesizer (Applied Biosystems). The oligonucleotide sequence of primers or probes may be derived from either strand of the duplex DNA. The primers or probes may consist of the bases A, G, C, or T or analogs and they may be degenerated at one or more chosen nucleotide position(s), using a nucleotide analog that pairs with any of the four naturally occurring nucleotides. (Nichols et al., 1994, Nature 369:492-493). Primers and probes may also contain nucleotide analogs such as Locked Nucleic Acids (LNA) (Koskin et al., 1998, Tetrahedron 54:3607-3630), and Peptide Nucleic Acids (PNA) (Egholm et al., 1993, Nature 365:566-568). Primers or probes may be of any suitable length, and may be selected anywhere within the DNA sequences of the bacterium to be detected, or from selected database sequences which are suitable for the detection of MRSA. In various embodiments, the primers and/or probes are at least 10, 12, 14, 16, 18, 20, 25, or 30 nucleotides in length.

[0048] Variants for a given target microbial gene are naturally occurring and are attributable to sequence variation within that gene during evolution. For example, different strains of the same microbial species may have a single or more nucleotide variation(s) at the oligonucleotide hybridization site. The skilled artisan readily appreciates the existence of variant nucleic acids and/or sequences for a specific gene and that the frequency of sequence variations depends on the selective pressure during evolution on a given gene product. Detection of a variant sequence for a region between two PCR primers may be achieved by sequencing the amplification product. Similar strategy may be used to detect variations at the hybridization site of a probe. Insofar as the divergence of the target nucleic acids and/or sequences or a part thereof does not affect significantly the sensitivity and/or specificity and/or ubiquity of the amplification primers or probes, variant MREJ sequences are contemplated, as are variant primer and/or probe sequences useful for amplification or hybridization to the variant MREJ.

[0049] Samples may include but are not limited to: any clinical sample or specimen, any environmental sample, any microbial culture, any microbial colony, any tissue, and any cell line, including but not limited to nose swabs, groin swabs, bacterial colonies, liquid cultures, expectorations, skin wounds, throat swabs, blood samples, contaminated surfaces and liquids.

[0050] 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 by successive thermal cycles allowing 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). Standard amplification protocols may be modified to improve nucleic acid amplification efficiency, including modifications to the reaction mixture. (Chakrabarti and Schutt, 2002, Biotechniques, 32:866-874; Al-Soud and Radstrom, 2002, 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 (TMAC).

[0051] It is also conceivable according to the present invention to use other types of nucleic acid amplification technology in the methods described herein. Non-limiting examples of amplification reactions that could be used in the methods described herein include but are not restricted to 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, MuIMs (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.

[0052] Although not used in the examples described hereinafter, it is possible to detect the amplicons produced by the amplification by using other well known methods. For example, the TaqMan® genotyping assay (Applied Biosystems, CA) combines hybridization and 5' nuclease activity of polymerase coupled with fluorescence detection. Other suitable detection methods are based on the use of molecular beacons. Molecular beacons (MBs) are oligonucleotide probes with complementary bases at either end so that the ends pair up to form a stem and loop structure. A donor and acceptor dye is attached to either end of the molecular beacon probe so that the probe suppresses fluorescence in its native conformation due to FRET. (See, Kramer F R., 1996, Nat Biotechnol 3:303-8.) For review of available technologies, see Sobin Kin and Ashish Misra, Annu. Rev. Biomed. Eng. 2007. 9 :7.1-7.32).

[0053] According to preferred embodiments of the invention, the detection of amplified nucleic acids is achieved using microspheres labelled with probes according to the Luminex® system. 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, including any real-time or post-amplification technologies known to those skilled in the art. Amplicon detection may also be performed by solid support or liquid hybridization using species-specific internal DNA probes hybridizing to an amplification product. Alternatively, amplicons can be characterized by sequencing. 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. No's: 5,866,366; 6,090,592; 6,117,635; and 6,117,986). 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 the nucleic acids of target genes.

[0054] The invention also concerns a kit to identify the presence of a methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample. A typical kit of the invention comprises amplification materials (e.g. primers, PCR positive controls) and detection materials (e.g. probes, microspheres), and the like

[0055] Kits of the invention can further comprises of or more of the following elements: buffers (e.g. lysis buffer, hybridization buffer, TE buffer), control DNA (e.g. bacterial genomic DNA), laboratory supplies (e.g. microfuge tubes, 96 well plates) and a user manual.

[0056] The diagnostic kits, primers and probes disclosed herein can be used to detect and/or identify antibiotic-resistant bacteria, and more particularly MRSA in both in vitro and/or in situ applications. For example, it is contemplated that the kits may be used in combination with previously described primers/probes detecting MRSA. 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.

[0057] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following examples, which should not be construed as further limiting. Examples

[0058] The examples set forth herein below provide exemplary methods for identifying the presence of a methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample. Also provided are exemplary protocols, molecular tools, probes, primers and techniques.

EXAMPLE 1 : General principles for the detection of SARM in a sample using PCR amplification and DNA-coupled microspheres probes

[0059] The general principles of the present invention are better understood by referring to the Figure 1. Figure 1 which illustrates an example of a multiplex PCR reaction for the simultaneous detection, in a single reaction tube, of the presence or absence of different strains of MRSA in a biological specimen from a patient (e.g. nose swab). In that figure, the horizontal arrows represent the primers used for the amplification of both (i) the SCCmec to the left of the integration and (ii) part of the orfX gene of MRSA. The black circles represent biotin molecules located at the 5'ends of the primers FMA3'bio and RXδ'bio. The double-lines "A", "B" and "C" represents the three PCR products that will be generated (assuming that the sample comprises only one type of MRSA among the different types that can be detected in that example). The white circles and adjacent lines represent specific DNA probes coupled to microspheres, those microspheres being later detected for confirming the presence or absence of a MRSA strain in the sample.

[0060] Figure 1 divides the genome of MRSA in two regions: the SCCmec region on the left (comprising the mecA gene) and the orfX gene on the right, both regions being divided by mecA's integration site which is referred hereinbelow as the "integration site". In this embodiment, the PCR is the preferred amplification method and up to height (8) different primers are being used for amplifying desired portion of S. aureus genome. As shown in Figure 1 , the first amplified region extends over the integration site and includes a portion of SSCmec and a portion of the orfX gene which is distinct from the portion of the first region. The second amplified region includes a part of the orfX gene. Those skilled in the art will readily understand that, according to the methods of the invention, it is preferable to avoid any overlap (or at least minimize the same) between the first and second region to be amplified. Indeed, an overlap may cause the complementary strands to hybridize together during the PCR amplification, thereby generating undesirable PCR products extending from one extremity of the first region to the second extremity of the second region.

[0061] Table 1 hereinafter provides the nucleotide sequence of each of the primers shown in Figure 1. More particularly, the primers identified as RMA1 , RMA2, RMA3, RMA4, and RMA5 are five different reverse primers (i.e. 5' -> 3') specific for the five known variable region of the S. aureus SCCmec element carrying mecA responsible for the methicillin resistance (stains of type i, ii, iii, iv and vi). The primer identified as FMA3'bio is a forward primer (i.e. 3' -> 5') which is specific for the orfX gene and is located around the integration site. As it will be described with more details hereinafter, this marker is further labelled with a biotin molecule (as evidenced by the presence of a black star). The seventh primer is identified as RXδ'bio. This primer is a reverse primer (i.e. 5' -> 3') which is specific for the strand orfX gene and which is complementary to the strand recognized by the primer FMA3'bio. The primer RXδ'bio is also labelled with a biotin marker (as evidenced by the presence of a star) and is also located around the integration site. The last primer is FX3'. This primer is a forward primer (i.e. 3' -> 5'), it is specific for the orfX gene and it is located at a certain distance away from the integration site, and more particularly, at a certain distance from the RXδ'bio primer.

[0062] Using those primers and the proper set of PCR conditions, specific regions of the S. aureus genome will be amplified, thereby resulting in the formation of "amplicons". In the embodiment illustrated in Figure 1 , three main categories of amplicons will be generated. The first category may comprises up to five (5) different amplicons that are generated by using the combination of the forward primer FMA3'bio and each of the reverse primers RMA1 , RMA2, RMA3, RMA4, and RMA5, each one of those amplicons overlapping the integration site and comprising both, a portion of the right extremity of SCCmec integration site (i.e. the extremity closest to the 5'end of orfX) and a portion of the orfX region. Since the FMA3'bio is biotinylated, those amplicons will also be biotinylated. Typically the biological sample will contain only one particular strain of MRSA and thus, only one replicon will be produced (i.e. the PCR product of any one of the reverse primers RMA1 to RMA5 and forward primer FMA3'bio), that amplicon being identified as "A" in Figure 1 ).

[0063] As shown also in the embodiment illustrated in Figure 1 , the reverse primer RXδ'bio will work in cooperation with the forward primer FX3' for generating an amplicon representative of a portion of the orfX region. In that particular example, the amplicons resulting from the amplification with the RXδ'bio and FX3' probes and those amplicons will be biotinylated because the primer RXδ'bio is biotinylated. Finally, the reverse primers RMA1 to RMAδ will work in cooperation with the forward primer FX3' for generating a possibility of up to five longer amplicons (according to the number of different MRSA strain in the sample). Those longer amplicons are not useful according to this embodiment and are ignored. None of those longer amplicons will interfere in the subsequent labelling and detection steps because, according to this preferred embodiment, those amplicons are being generated using unlabelled primers and are therefore not detected in future steps. The size of the amplicons or PCR products generated using the specific primers described herein is given in Table 2.

[0064] Needless to say that the amplification reactions described above could also be carried out in parallel in separate vessels instead of being run in a multiplex format. For instance it is possible to run separate amplification reactions for each combination of RMA primers/ FMA3'bio. Similarly, the amplification of the orfX region (RXδ'bio/FX3' combination) could be run in a separate vessel. Thereafter, the PCR products of those multiple separate reactions could be further evaluated separately or pooled together for the detection step.

[0065] The above example refers to biotin tagged primers. The biotin is a molecule which is well known for its affinity for the bacterial protein streptavidin. As described herein after, this affinity is being used in preferred embodiments according to the invention for invention for binding avidin-coupled chromophores to the amplicons which will be later detected. Those skilled in the art will understand that other combination of binding molecules can be used according to the principles of the invention.

[0066] In the preferred embodiments, the invention relies on the use of probes for the detection of the two amplicons. Those probes comprise of a nucleic acid molecule (typically DNA) coupled to microspheres, this nucleic acid molecule having a sequence capable of hybridizing specifically to the amplicon to be detected. Table 1 hereinafter provides the sequence of two preferred probes, namely OXSAS-2 which hybridizes a region of orfX close to the integration site and MASS-2 which hybridizes to a different region of orfX (see also Figure 1 for approximate positions).

[0067] According to that embodiment, the making of the probes requires a modification at the 5' extremity of the nucleic acid molecule, this modification being of the type "5'-Uni-link amino". This modification is necessary for coupling the nucleic acid molecule to the microspheres, but it is conceivable that other coupling method, strategies and modification may achieve the same purpose, including but not limited to 5' amino modifier C6 or C12.

[0068] Suitable microspheres according to the invention typically have a diameter between few microns and 1 mm. The microspheres preferred according to the invention are commercially available from Luminex Corporation, Qiagen,

Biorad and others. Those microspheres are made of polystyrene and are internally dyed with two spectrally distinct fluorochromes for detection using the

Luminex® microspheres technology (see Dunbar S. Clinica Chimica Acta, 363 (2006) 71-82 which is incorporated herein in its entirety). Using precise amounts of each of these fluorochromes, an array is created consisting of 100 different microsphere sets with specific spectral address. Since each microsphere sets can possess a different reactant surface, the preferred embodiments of the invention use that property for making the two desired probes by coupling the two nucleic acid molecules referred to hereinbefore. Thereafter the probes are hybridized to the amplicons thereby making new complexes comprising the probes and the amplicons. Those complexes can be recovered by centrifugation and resuspended in a solution containing a reporter molecule.

[0069] According to that embodiment, the reporter molecule comprises two elements: a first element having a strong affinity for the ligand present on the primers and on the resulting amplicons. In the case of the biotin marker, the preferred first element is the streptavidin. The second element is bound to the first element. The second element is another chromophore having an emitting wave length different from the wavelength of the dyes in the microspheres. Various chromophores can be used but the one preferred according to the invention is R-phycoerythrin (known also as "Alexa 532" or "Cy3") and it is excited at 532 nm, 13 mW with a yttrium aluminum garnet (YAT) laser. During normal operation, microspheres are interrogated individually in a rapidly flowing fluid stream as they pass by two separate lasers in the Luminex® 100™ analyzer. A 635-nm 10-mW red diode laser excites the two fluorochromes contained within the microspheres and the yttrium aluminum garnet laser excites the reporter fluorochrome (i.e. R-phycoerythrin). High speed digital signal processing classifies the microsphere based on its spectral address and quantifies the reaction on the surface. Thousands of microspheres are interrogated per second resulting in an analysis system capable of analyzing and reporting up to 100 different reactions in a single reaction vessel in just a few seconds per sample. As indicated hereinbefore, the embodiments of invention described in details herein utilizes only two different sets of microspheres (one set for each amplicon to be detected) but it is conceivable to expand capabilities of the present invention by using more than two sets of microspheres (e.g. internal PCR positive control, one set of microsphere for each different strain or subtype of MRSA, etc.). EXAMPLE 2: Optimization of a multiplex PCR-based method for the detection of SARM

Step 1 : Preparation of the specimens

[0070] The specimens may be from various origins: nasal swabs, bacterial colonies, blood samples, expectorations, skin wounds, bacterial cultures, etc. The specimen is either directly resuspended into 25 μl_ of lysis buffer or spun into a microfuge tube and the pellet is resuspended by vortex into 25 μl_ of lysis buffer.

The lysis buffer is composed of (but not restricted to) : Triton X-100 at 1 %, Tween

20 at 0.5%, Tris-HCI (pHβ.O) at 10 mM and EDTA (pHβ.O) at 1 mM final concentration. The sample is heated at 95⁰C for 10 minutes and spun at >8000 x g for 5 minutes. The supernatant is collected and transferred into a cleaned tube.

Step 2. The PCR reaction [0071] The PCR reaction is set as follow : Master mix volumes final concentration

Water 16.8 μL

10X buffer 2.5 μL 1x

25 mM dNTPs 0.25 μL 250 μM Oligonucleotide mix 0.25 μL RMA1 ,2,3,4,5 FX3' at 0.1 mM

FMA3'bio and RXδ'bio at 0.2 mM Taq polymerase 0.20 uL 1 unit

20 μL

Table 1 : Primers and probes for the PCR reaction

Table 2: PCR products

[0072] An amount of 5.0 μl_ of the specimen is added to the 20 μL of master mix and put into a thermocycler programmed to perform the following steps. Thermocycling conditions:

Steps T f O) Time

1 95° C 10 min

2 94° C 30 sec

3 60° C 40 sec

4 72° C 1 min (repeat step 2 to 4 44 times)

5 72° C 5 min

6 4°C pause

Step 3. Detection with the Luminex®

[0073] This detection step is carried out essentially as described by Sherry A. Dunbar and James W. Jacobson in Methods in Molecular Medicine, vol. 144, Microarrays in Clinical Diagnostics, T. Joos and P. Fortine Eds, Humana Press Inc., Totowa, NJ.

[0074]

1. The microspheres coupled to the probes OXSAS2 and those coupled to the probes MASS2 are resuspended by vortex for 20 seconds and by sonication for 20 seconds. The microspheres are than diluted onto 1.5X

TMAC buffer to a final concentration of 1500 microspheres per reaction.

2. In a 96 well plate, the first series is comprised of : 10 μl_ of TE buffer, 25 μl_ of the microsphere mix and than, add 2.5 μl_ of the PCR reaction. The second series is comprised of 7.5 μl_ of TE buffer, 25 μl_ of the microsphere mix and than, add 5.0 μL of the PCR reaction.

3. The 96 well plate is sealed and placed in a thermocycler. Heat at 95°C for 5 minutes and hybridize at 57°C for 15 minutes.

4. The plate is rapidly spun at 3000 x g for 5 minutes and the supernatant is removed by flipping the plate 3 times over the sink. 5. Add 75 μL of 1 X TMAC buffer containing 4 μg/mL of SAPE and resuspend the pellets 15 times.

6. Incubate in the dark for 10 minutes.

7. Program the Luminex in order to detect a minimum of 100 microspheres for each sets in a time of 45 seconds and a volume of 50 μL. 8. At the end of the run, compare the median fluorescence intensity obtained with the 2.5 μL PCR samples with the 5.0 μL PCR samples.

[0075] Figure 2 shows the median fluorescence intensity obtained with increasing amounts of orfX PCR product. The orfX region was amplified using

RXδ'bio and FX3' oligos using S. aureus genomic DNA (Black Bars) or a no- template negative control (White Bars). Various amounts of the PCR reaction ranging from 0.015 μl_ up to 5.0 μl_ were hybridized to the OXSAS2 captured probe coupled to a microsphere set and the median fluorescence intensity was measured using the microsphere-based suspension platform from Luminex® (Luminex Corporation, Austin, Texas). As shown, increasing amount of the PCR product from S. aureus resulted in a direct increase in the mean fluorescence intensity.

[0076] Figure 3 shows the median fluorescence intensity obtained with increasing amounts of the mecA/orfX junction PCR product. The mecA/orfX junction region was amplified using the RMA1 , RMA2, RMA3, RMA4, RMA5 and FMA3'bio oligos using MRSA strain 43300 genomic DNA (Black Bars) or a no- template negative control (White Bars). Various amounts of the PCR reaction ranging from 0.015 μl_ up to 5.0 μl_ was hybridized to the MASS2 captured probe coupled to a microsphere set and the median fluorescence intensity was measured using the microsphere-based suspension platform from Luminex (Luminex Corporation, Austin, Texas). As shown, increasing amount of the mecA/orfX junction PCR product resulted in a direct increase in the mean fluorescence intensity.

[0077] Figure 4 shows the median fluorescence intensity (MFI) obtained with MRSA negative and MRSA positive specimens. Various specimens comprised of C- (lysis buffer only), C+ (MRSA genomic DNA), various nasal specimens from hospitalized patients (#86, 87, 88, and 89) as well as S. aureus strain 25923 (MRSA negative) were used to amplify the orfX and mecA/orfX junction region by duplex PCR. An aliquot of 2.5 μL and 5.0 μL were hybridized to the OXSAS2 captured probe coupled microspheres set 1 and MASS2 captured probe coupled microspheres set 2. The white bars are indicative of the presence of S. aureus {orfX) and the black bars are indicative of the MRSA (mecAlorfX junction). For each sample the first two bars (white:black) represent the aliquot of 2.5 μL whereas the third and fourth bars (white:black) represent the 5.0 μL aliquot. [0078] As shown in this Figure, there is a significant increase the MFI associated with the detection of the orfX gene (White Bars) for the specimens C+ (MRSA positive control), specimen #86 and S. aureus strain 25923. This result confirms that those three specimens comprise S. aureus. However, when measuring the MFI resulting from two quantities of the PCR product from the amplification of the mecA/orfX junction (Black Bars), only the MFI of specimens C+ and #86 is increased. This confirms that both the C+, the specimen #86 are MRSA positive and that the S. aureus specimen (strain 25923) is MRSA negative, as expected. The overall conclusion is that the controls responded as expected and that, among the four clinical specimens analyzed, only specimen #86 contains S. aureus and that strain of S. aureus is methicillin resistant.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

1. A method to identify the presence of a methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample, said MRSA strain having a genome with a SCCmec inserted at an integration site in said genome, said method comprising: amplifying a first region of the genome of said strain, said first region comprising said integration site and extending from a first position in said SCCmec to a second position in said genome; amplifying a second region of the genome of said strain, said second region being specific to S. aureus and extending from third and forth positions in said genome, wherein the second, third and a forth positions are selected for priming under suitable amplification conditions the production of two distinct amplicons corresponding to said first and second region; identifying the presence of said MRSA strain in said sample by detecting the presence of both amplicons.

2. The method of claim 1 , wherein said second, third and a forth positions are selected such that the amplified second region does not overlap the amplified first region.

3. The method of claim 1 or 2, wherein said first, second, third and a forth positions are selected such that said amplicons comprises about 50 bp to about 20 kb.

4. The method of any one of claims 1 to 3, wherein said genome comprises an orfX gene and wherein said second position is located inside the orfX gene.

5. The method of any one of claims 1 to 3, wherein said genome comprises an or/X gene and wherein said second region corresponds to a part of the orfX gene.

6. The method of any one of claims 1 to 5, wherein amplification of said first region and second region is carried out in a single reaction vessel.

7. The method of any one of claims 1 to 6, wherein the amplification of said first region and second region is carried out by using a technique of polymerase chain reaction.

8. The method of any one of claims 1 to 7, wherein detection of each amplicon comprises using at least two different quantities thereof, measuring for each one of said at least two quantities a detection signal, and comparing said detection signals, whereby a reliable difference in said detection signals is indicative of the presence of the amplicon to be detected.

9. The method of any one of claims 1 to 8, wherein the detection of said two amplicons comprises hybridizing thereto first and second labelled probes specific for said first region and second region, respectively.

10. The method of claim 9, wherein each labelled probe comprises of a nucleic acid molecule that hybridizes specifically to the amplicon to be detected, said nucleic acid molecule being coupled to microspheres.

11. The method of claim 10, wherein said microspheres are dyed with two spectrally distinct fluorochromes for detection using the Luminex® microspheres technology.

12. A method to identify the presence of a methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample, said MRSA strain comprising a genome with a SCCmec inserted at an integration site in said genome, said method comprising: using a set of primers for amplifying first and second regions of said genome, the first region comprising said integration site and extending from a first position in said SCCmec to a second position in said genome; the second region being specific to S. aureus and extending from third and forth positions in said genome, wherein said primers are selected for priming under suitable amplification conditions the production of two distinct amplicons of about 50 bp to about 20 kb corresponding to said first and second region, and; hybridizing to said amplicons first and second labelled probes specific for said first and second region, respectively; detecting the labelled probes hybridized to a corresponding amplicon, wherein detection of each of the hybridized labelled probes comprises using at least two different quantities thereof, measuring for each one of said at least two quantities a detection signal, and comparing said detection signals, wherein a reliable difference in said detection signals is indicative of the presence of the amplicon to be detected; whereby the presence of both of said amplicons is indicative of the presence of said MRSA strain in said sample.

13. The method of claim 12, wherein said the labelled probes consists of nucleic acid-coupled microspheres suitable for detection using the Luminex® technology.

14. The method of claim 12 or 13, wherein the primers for the first position in the first region comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

15. The method of any one of claims 12 to 14, wherein the primer for the second position in the first region comprises a nucleic acid sequence as set forth in SEQ ID NO:8.

16. The method any one of claims 12 to 14, wherein the primer for the third position in the second region comprises a nucleic acid sequence as set forth in SEQ ID NO:1.

17. The method one of claims 12 to 14, wherein the primer for the forth position in the second region comprises a nucleic acid sequence as set forth in SEQ ID NO:2.

18. A kit to identify the presence of methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample, said MRSA strain comprising a genome with a SCCmec inserted at an integration site in said genome, the kit comprising: primers for amplifying first and second regions of said genome, the first region comprising said integration site and extending from a first position in said SCCmec to a second position in said genome; the second region being specific to S. aureus and extending from third and forth positions in said genome; wherein said primers are selected for priming under suitable amplification conditions the production of two distinct amplicons of about 50 bp to about 20 kb corresponding to said first and second regions.

19. The kit of claim 18, wherein said genome comprises an orfX gene and wherein said primers are selected for the amplification of said first and second regions by using a technique of polymerase chain reaction, said kit comprising:

- a first set of primers for priming the production of the first amplicon, said first set comprising at least one primer hybridizing to SCCmec and one primer hybridizing to a position located inside the orfX gene;

- a second set of primers for priming the production of the second amplicon, said second set comprising two primers hybridizing to the orfX gene;

- first and second probes hybridizing specifically to said first and second amplicons, respectively.

20. The kit of claim 19, wherein the primer hybridizing to SCCmec comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 and wherein the primer hybridizing to a position is located inside the orfX gene comprises a nucleic acid sequence as set forth in SEQ ID NO:8.

21. The kit of claim 19 or 20, wherein the second set of primers comprises a nucleic acid sequence selected from SEQ ID NO:1 and SEQ ID NO:2.

22. The kit of any one of claims 19 to 21 , wherein the first and second probes comprises a nucleic acid sequence selected from SEQ ID NO:9 and SEQ ID NO:10.

23. A method to identify the presence of a methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample, said MRSA strain comprising a genome with a SCCmec inserted at an integration site in said genome, said method comprising:

- amplifying a first region of the genome of the MRSA strain, the first region including said integration site and extending on both sides thereof;