description lang="EN" 
BACKGROUND OF THE INVENTION 

 Clinical significance ofStaphylococcus aureus 

[0001] The coagulase-positive speciesStaphylococcus aureusis well documented as a human opportunistic pathogen. Nosocomial infections caused byS.aureusare a major cause of morbidity and mortality. Some of the most common infections caused byS. aureusinvolve the skin, and they include furuncles or boils, cellulitis, impetigo, and postoperative wound infections at various sites. Some of the more serious infections produced byS. aureusare 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 withS:aureus.Toxic shock syndrome, a community-acquired disease, has also been attributed to infection or colonization with toxigenicS.aureus( Murray et al. Eds, 1999, Manual of Clinical Microbiology, 7th Ed., ASM Press, Washington, D.C .). 

[0002] Methicillin-resistantS. aureus(MRSA) emerged in the 1980s as a major clinical and epidemiologic problem in hospitals. MRSA are resistant to all β-lactams including penicillins, cephalosporins, carbapenems, and monobactams, which are the most commonly used antibiotics to cureS.aureusinfections. MRSA infections can only be treated with more toxic and more costly antibiotics, which are normally used as the last line of defence. 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. 

[0003] Methicillin resistance inS. aureusis 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 toβ-lactam antibiotics. S.aureusnormally 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 choromosomalmecAgene and functions as a β-lactam-resistant transpeptidase. ThemecAgene is absent from methicillin-sensitiveS.aureusbut is widely distributed among other species of staphylococci and is highly conserved ( Ubukata et al., 1990, Antimicrob. Agents Chemother. 34:170-172 ). 

[0004] By nucleotide sequence determination of the DNA region surroundingthemecAgene fromS.aureusstrain N315 (isolated in Japan in 1982), Hiramatsuet al.have found that themecAgene is carried by a novel genetic element, designated staphylococcal cassette chromosomemec(SCCmec),inserted into the chromosome. SCCmecis a mobile genetic element characterized by the presence of terminal inverted and direct repeats, a set of site-specific recombinase genes (ccrAandccrB),and themecAgene complex ( Ito et al., 1999, Antimicrob. Agents Chemother. 43:1449-1458 ; Katayama et al., 2000, Antimicrob. Agents Chemother. 44:1549-1555 ). The element is precisely excised from the chromosome of S.aureusstrain N315 and integrates into a specificSaureuschromosomal site in the same orientation through the function of a unique set of recombinase genes comprisingccrAandccrB.Two novel genetic elements that shared similar structural features ofSCCmecwere found by cloning and sequencing the DNA region surrounding themecAgene 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). The threeSCCmechave 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 ) ( Figure 1 ). Hiramatsuet al.have found that theSCCmecDNAs are integrated at a specific site in the methicillin-sensitiveS.aureus(MSSA) chromosome. They characterized the nucleotide sequences of the regions around the left and right boundaries ofSCCmecDNA (i.e.attL andattR, respectively) as well as those of the regions around theSCCmecDNA integration site (i.e.attBsccwhich is the bacterial chromosome attachment site forSCCmecDNA). Theattbsccsite was located at the 3' end of a novel open reading frame (ORF),orfX.TheorfXpotentially encodes a 159-amino acid polypeptide sharing identity with some previously identified polypeptides, but of unknown function ( Ito et al., 1999, Antimicrob. Agents Chemother. 43:1449-1458 ). Recently, a new type ofSCCmec(type IV) has been described by both Hiramatsuet al.( Ma al, 2002, Antimicrob. Agents Chemother. 46:1147-1152 ) and Oliveiraet al.( Oliveira et al, 2001, Microb. Drug Resist. 7:349-360 ). The sequences of the right extremity of the new type IVSCCmecfromS. aureusstrains CA05 and 8/6-3P published by Hiramatsuet al. ,( Ma et al., 2002, Antimicrob. Agents Chemother. 46:1147-1152 ) were nearly identical over 2000 nucleotides to that of type II SCCmecof Saureusstrain N315 ( Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-1336 ). No sequence at the right extremity of the SCCmectype IV is available from theS. aureusstrains HDE288 and PL72 described by Oliveiraet al.( Oliveira et al., 2001, Microb.Drug Resist. 7;349-360 ). 

[0005] Previous methods used to detects and identify MRSA ( Saito et al., 1995, J. Clin. Microbiol. 33:2498-2500 ; Ubukataet 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 ), which are based on the detection of themecAgene andS. aureus-specific chromosomal sequences, encountered difficulty in discriminating MRSA from methicillin-resistant coagulase-negative staphylococci (CNS) because themecAgene is widely distributed in both S.aureusand CNS species ( Suzuki et al., 1992, Antimicrob. Agents. Chemother. 36:429-434 ). Hiramatsu et al. (US patent 6,156,507 ) have described a PCR assay specific for MRSA by using primers that can specifically hybridize to the right extremities of the 3 types of SCCmecDNAs in combination with a primer specific to the S.aureuschromosome, which corresponds to the nucleotide sequence on the right side of theSCCmecintegration site. Since nucleotide sequences surrounding theSCCmecintegration site in other staphylococcal species (such asS. epidermidisand S.haemolyticus) are different from those found inS. aureus,this PCR assay was specific for the detection of MRSA. This PCR assay also supplied information for MREP typing (standing for«mecright extremity polymorphism») ofSCCmecDNA ( Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-1336 ; Hiramatsu et al., 1996, J. Infect. Chemother. 2:117-129 ). This typing method takes advantage of the polymorphism at the right extremity of SCCmecDNAs adjacent to the integration site among the three types of SCCmec.Type III has a unique nucleotide sequence while type II has an insertion of 102 nucleotides to the right terminus ofSCCmectype I. The MREP typing method described by Hiramatsuetal.( Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-1336 ; Hiramatsu et al., 1996, J. Infect. Chemother. 2:117-129 ) defines the SCCmectype I as MREP type i,SCCmectype II as MREP type ii and SCCmectype III as MREP type iii. It should be noted that the MREP typing method cannot differentiate the new SCCmectype IV described by Hiramatsuet al.( Ma et al., 2002, Antimicrob. Agents Chemother. 46:1147-1152 ) from SCCmectype II because these two SCCmectypes exhibit the same nucleotide sequence to the right extremity. 

[0006] The set of primers described by Hiramatsu et al. as being the optimal primer combination (SEQ ID NOs.: 22, 24, 28 in US patent 6,156,507 corresponding to SEQ ID NOs.: 56, 58 and 60, respectively, in the present invention) have been used in the present invention to test by PCR a variety of MRSA and MSSA strains ( Figure 1 and Table 1). Twenty of the 39 MRSA strains tested were not amplified by the Hiramatsu et al. multiplex PCR assay (Tables 2 and 3). Hiramitsu's method indeed was successful in detecting less than 50% of the tested 39 MRSA strains. This finding demonstrates that some MRSA strains have sequences at the right extremity of SCCmec-chromosome right extremity junction different from those identified by Hiramatsuet al.Consequently, the system developed by Hiramatsuet,al.does not allow the detection of all MRSA. The present invention relates to the generation of SCCmec-chromosome right extremity junction sequence data required to detect more MRSA strains in order to improve the Hiramatsuet al.assay. There is a need for developing more ubiquitous primers and probes for the detection of most MRSA strains around the world. 

 SUMMARY OF THE INVENTION 

[0007] It is an object of the present invention to provide a specific, ubiquitous and sensitive method using probes and/or amplification primers for determining the presence and/or amount of nucleic acids from all MRSA strains. 

[0008] Ubiquity of at least 50% amongst the strains representing MRSA strains types IV to X is an objective of this invention. 

[0009] Therefore, in accordance with the present invention is provided a method to detect the presence of a methicillin-resistantStaphylococcus aureus(MESA) strain in a sample, the MRSA strain being resistant because of the presence of anSCCmecinsert containing a mecA gene, saidSCCmecbeing inserted in bacterial nucleic acids thereby generating a polymorphic right extremity junction (MREJ), the method comprising the step of annealing the nucleic acids of the sample with a plurality of probes and/or primers, characterized by:
(i) the primers and/or probes are specific for MRSA strains and capable of annealing with polymorphic MREJ nucleic acids, the polymorphic MREJ comprising MREJ types i to x; and
(ii) the primers and/or probes altogether can anneal with at least four MREJ types selected from MREJ types i to x.

[0010] In a specific embodiment, the primers and/or probes are all chosen to anneal under common annealing conditions, and even more specifically, they are placed altogether in the same physical enclosure. 

[0011] A specific method has been developed using primers and/or probes having at least 10 nucleotides in length and capable of annealing with MREJ types i to iii, defined in any one of SEQ ID NOs: 1, 20, 21, 22, 23, 24,25, 41,199 ; 2, 17, 18, 19, 26, 40, 173; 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 197 ; 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 104, 184, 198 and with one or more of MREJ types iv to ix, having SEQ ID NOs: 42, 43, 44, 45,46, 51 ; 47, 48, 49, 50 ; 171 ; 165, 166 ; 167 ; 168. To be perfectly ubiquitous with the all the sequenced MREJs, the primers and/or probes altogether can anneal with said SEQ ID NOs of MREJ types i to ix. 

[0012] The following specific primers and/or probes having the following sequences have been designed:
<tb>66, 100, 101, 105, 52, 53, 54, 55,for the detection of MREJ type i
<tb>56,57,64,71,72,73,74,75,76,
<tb>70, 103, 130, 132, 158, 159, 59,
<tb>62, 126, 127, 128, 129, 131, 200,
<tb>201,60,61,63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>66, 97, 99, 100, 101, 106, 117,for the detection of MREJ type ii
<tb>118, 124, 125, 52, 53, 54, 5, 56, 57
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 32, 58, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60, 61, 63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>67, 98, 102, 107, 108for the detection of MREJ type iii
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 132, 158, 159
<tb>58,
<tb>59, 62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60, 61, 63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>79, 77, 145, 147for the detection of MREJ type iv
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60, 61, 63
<tb>68
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>65, 80, 146, 154, 155 forthe detection of MREJ type v
<tb>64, 71, 72, 73, 74, 75, 76,
<tb>70, 103, 130, 132, 158, 159
<tb>59, 62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60, 61, 63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>202, 203, 204for the detection of MREJ type vi
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60, 61, 63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>112, 113, 114, 119, 120, 121, 122for the detection of MREJ type vii,
<tb>123, 150, 151, 153
<tb>64, 71, 72, 73, 74, 75, 76, 70, 103,
<tb>130, 132, 158, 159
<tb>59, 62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60, 61, 63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>115, 116, 187, 188, 207, 208 forthe detection of MREJ type viii
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60,61,63
<tb>32,83,84,160,161,162,163,164
<tb>85, 86, 87, 88, 89
<tb>109, 148, 149, 205, 206for the detection of MREJ type ix.
<tb>64, 71, 72, 73, 74, 75, 76
<tb>70, 103, 130, 132, 158, 159
<tb>59, 62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60, 61, 63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89

[0013] Amongst these, the following primer pairs having the following sequences are used:
<tb>64/66, 64/100, 64/101; 59/52,for the detection of type i MREJ
<tb>59/53, 59/54, 59/55, 59/56, 59/57,
<tb>60/52, 60/53, 60/54, 60/55, 60/56
<tb>60/57, 61/52, 61/53, 61/54, 61/55
<tb>61/56, 61/57, 62/52, 62/53, 62/54
<tb>62/55, 62/56, 62/57, 63/52, 63/53
<tb>63/54, 63/55, 63/56, 63/57
<tb>64/66, 64/97, 64/99, 64/100, 64/101for the detection of type ii MREJ,
<tb>59/52, 59/53, 59/54, 59/55, 59/56,
<tb>59/57, 60/52, 60/53, 60/54, 60/55,
<tb>60/56, 60/57, 61/52, 61/53, 61/54,
<tb>61/55, 61/56, 61/57, 62/52, 62/53,
<tb>62/54, 62/55, 62/56, 62/57, 63/52
<tb>63/53, 63/54, 63/55, 63/56, 63/57
<tb>64/67, 64/98, 64/102 ; 59/58,for the detection of type iii MREJ
<tb>60/58, 61/58, 62/58, 63/58
<tb>64/79for the detection of type iv MREJ
<tb>64/80for the detection of type v MREJ
<tb>64/204for the detection of type vi MREJ
<tb>64/112, 64/113for the detection of type vii MREJ
<tb>64/115, 64/116for the detection of type viii MREJ
<tb>64/109for the detection of type ix MREJ

[0014] As well, amongst these, the following probes having the following sequences are used:
SEQ ID NOs: 32, 83, 84, 160, 161, 162, 163, 164 for the detection of MREJ types i to ix.

[0015] In the most preferred embodied method, the following primers and/or probes having the following nucleotide sequences are used together. The preferred combinations make use of:
i) SEQ ID NOs: 64, 66, 84,163,164 for the detection of MREJ type i
ii) SEQ ID NOs: 64, 66, 84, 163, 164 for the detection of MREJ type ii
iii) SEQ ID NOs: 64, 67, 84, 163, 164 for the detection of MREJ type iii
iv) SEQ ID NOs: 64, 79, 84, 163, 164 for the detection of MREJ type iv
v) SEQ ID NOs: 64, 80, 84, 163, 164 for the detection of MREJ type v
vi) SEQ ID NOs: 64, 112, 84, 163, 164 for the detection of MREJ type vii.

[0016] All these probes and primers can even be used together in the same physical enclosure. 

[0017] It is another object of this invention to provide a method for typing a MREJ of a MRSA strain, which comprises the steps of reproducing the above method with primers and/or probes specific for a determined MREJ type, and detecting an annealed probe or primer as an indication of the presence of a determined MREJ type. 

[0018] It is further another object of this invention to provide a nucleic acid selected from SEQ ID NOs:
i) SEQ ID NOs: 42, 43, 44, 45, 46, 51 for sequence of MREJ type iv ;
ii) SEQ ID NOs: 47, 48, 49, 50 for sequence of MREJ type v ;
iii) SEQ ID NOs: 171 for sequence of MREJ type vi ;
iv) SEQ ID NOs: 165, 166 for sequence of MREJ type vii ;
v) SEQ ID NOs: 167 for sequence of MREJ type viii ;
vi) SEQ ID NOs: 168 for sequence of MREJ type ix.

[0019] Oligonucleotides of at least 10 nucleotides in length which hybridize with any of these nucleic acids and which hybridize with one or more MREJ of types selected from iv to ix are also objects of this invention. Amongst these, primer pairs (or probes) having the following SEQ ID NOs:
<tb>64/66, 64/100, 64/101; 59/52,for the detection of type i MREJ
<tb>59/53, 59/54, 59/55, 59/56, 59/57,
<tb>60/52, 60/53, 60/54, 60/55, 60/56
<tb>60/57, 61/52, 61/53, 61/54, 61/55
<tb>61/56, 61/57, 62/52, 62/53, 62/54
<tb>62/55, 62/56, 62/57, 63/52, 63/53
<tb>63/54, 63/55, 63/56, 63/57
<tb>64/66, 64/97, 64/99, 64/100, 64/101for the detection of type ii MREJ
<tb>59/52, 59/53, 59/54, 59/55, 59/56,
<tb>59/57, 60/52, 60/53, 60/54, 60/55,
<tb>60/56, 60/57, 61/52, 61/53, 61/54,
<tb>61/55, 61/56, 61/57, 62/52, 62/53,
<tb>62/54, 62/55, 62/56, 62/57, 63/52
<tb>63/53, 63/54, 63/55, 63/56, 63/57
<tb>64/67, 64/98, 64/102 ; 59/58,for the detection of type iii MREJ
<tb>60/58, 61/58, 62/58, 63/58
<tb>64/79for the detection of type iv MREJ
<tb>64/80for the detection of type v MREJ
<tb>64/204for the detection of type vi MREJ
<tb>64/112, 64/113for the detection of type vii MREJ
<tb>64/115, 64/116for the detection of type viii MREJ
<tb>64/109for the detection of type ix MREJ,are also within the scope of this invention. 

[0020] Further, internal probes having nucleotide sequences defined in any one of SEQ ID NOs: 32, 83, 84, 160, 161, 162, 163, 164, are also within the scope of this invention. Compositions of matter comprising the primers and/or probes annealing or hybridizing with one or more MREJ of types selected from iv to ix as well as with the above nucleic acids, comprising or not primers and/or probes, which hybridize with one or more MREJ of types selected from i to iii, are further objects of this invention. The preferred compositions would comprise the primers having the nucleotide sequences defined in SEQ ID NOs:
<tb>64/66,64/100, 64/101; 59/52,for the detection of type i MREJ
<tb>59/53, 59/54, 59/55, 59/56, 59/57,
<tb>60/52, 60/53, 60/54, 60/55, 60/56
<tb>60/57,61/52, 61/53, 61/54, 61/55
<tb>61/56, 61/57, 62/52, 62/53, 62/54 '
<tb>62/55,62/56, 62/57, 63/52, 63/53
<tb>63/54, 63/55, 63/56, 63/57
<tb>64/66, 64/97, 64/99, 64/100, 64/101for the detection of type ii MREJ
<tb>59/52, 59/53, 59/54, 59/55, 59/56,
<tb>59/57, 60/52, 60/53, 60/54, 60/55,
<tb>60/56, 60/57, 61/52, 61/53, 61/54,
<tb>61/55,61/56, 61/57, 62/52, 62/53,
<tb>62/54, 62/55, 62/56, 62/57, 63/52
<tb>63/53, 63/54, 63/55, 63/56, 63/57
<tb>64/67, 64/98, 64/102 ; 59/58,for the detection of type iii MREJ
<tb>60/58, 61/58, 62/58, 63/58
<tb>64/79for the detection of type iv MREJ
<tb>64/80for the detection of type v MREJ
<tb>64/204for the detection of type vi MREJ
<tb>64/112, 64/113for the detection of type vii MREJ
<tb>64/115, 64/116for the detection of type viii MREJ
<tb>64/109for the detection of type ix MREJ,or probes, which SEQ ID NOs are: 32, 83, 84, 160, 161, 162, 163, 164, or both. 

 DETAILED DESCRIPTION OF THE INVENTION 

[0021] Here is particularly provided a method wherein each of MRSA nucleic acids or a variant or part thereof comprises a selected target region hybridizable with said primers or probes developed to be ubiquitous;
wherein each of said nucleic acids or a variant or part thereof comprises a selected target region hybridizable with said primers or probes ;
said method comprising the steps of contacting said sample with said probes or primers and detecting the presence and/or amount of hybridized probes or amplified products as an indication of the presence and/or amount of MRSA. 

[0022] In the method, sequences from DNA fragments of SCCmec-chromosome right extremity junction, therafter named MREJ standing for« mecright extremity junction » including sequences from SCCmecright extremity and chromosomal DNA to the right of the SCCmecintegration site are used as parental sequences from which are derived the primers and/or the probes. MREJ sequences include our proprietary sequences as well as sequences obtained from public databases and from US patent 6,156,507 and were selected for their capacity to sensitively, specifically, ubiquitously and rapidly detect the targeted MRSA nucleic acids. 

[0023] Our proprietary DNA fragments and oligonucleotides (primers and probes) are also another object of this invention. 

[0024] Composition of matters such as diagnostic kits comprising amplification primers or probes for the detection of MRSA are also objects of the present invention. 

[0025] In the above methods and kits, probes and primers are not limited to nucleic acids and may include, but are not restricted to, analogs of nucleotides. The diagnostic reagents constitued by the probes and the primers may be present in any suitable form (bound to a solid support, liquid, lyophilized, etc.). 

[0026] In the above methods and kits, amplification reactions may include but are not restricted to: a) polymerase chain reaction (PCR), b) ligase chain reaction (LCR), c) nucleic acid sequence-based amplification (NASBA), d) self-sustained sequence replication (3SR), e) strand displacement amplification (SDA), f) branched DNA signal amplification (bDNA), g) transcription-mediated amplification (TMA), h) cycling probe technology (CPT), i) nested PCR, j) multiplex PCR, k) solid phase amplification (SPA), 1) nuclease dependent signal amplification (NDSA), m) rolling circle amplification technology (RCA), n) Anchored strand displacement amplification, o) Solid-phase (immobilized) rolling circle amplification. 

[0027] In the above methods and kits, detection of the nucleic acids of target genes may include real-time or post-amplification technologies. These detection technologies can include, but are not limited to fluorescence resonance energy transfer (FRET)-based methods such as adjacent hybridization of probes (including probe-probe and probe-primer methods),TaqMan probe, molecular beacon probe, Scorpion probe, nanoparticle probe and Amplifluor probe. 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 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. 

[0028] In a preferred embodiment, a PCR protocol is used for nucleic acid amplification. 

[0029] A method for detection of a plurality of potential MRSA strains having different MREJ types may be conducted in separate reactions and physical enclosures, one type at the time. Alternatively, it could be conducted simultaneously for different types in separate physical enclosures, or in the same physical enclosures. In the latter scenario a multiplex PCR reaction could be conducted which would require that the oligonucleotides are all capable of annealing with a target reagion under common conditions. Since many probes or primers are specific for a determined MREJ type, typing a MRSA strain is a possible embodiment. When a mixture of oligonucleotides annealing together with more than one type is used in a single physical enclosure or container, different labels would be used to distinguish one type from another. 

[0030] We aim at developing a DNA-based test or kit to detect and identify MRSA. Although the sequences fromorfXgenes and someSCCmecDNA fragments are available from public databases and have been used to develop DNA-based tests for detection of MRSA, new sequence data allowing to improve MRSA detection and identification which are object of the present invention have either never been characterized previously or were known but not shown to be located at the right extremity ofSCCmecadjacent to the integration site (Table 4). These novel sequences could not have been predicted nor detected by the MRSA-specific PCR assay developed by Hiramatsu et al. (US patent 6,156,507 ). These sequences will allow to improve current DNA-based tests for the diagnosis of MRSA because they allow the design of ubiquitous primers and probes for the detection and identification of more MRSA strains including all the major epidemic clones from around the world. 

[0031] The diagnostic kits, primers and probes mentioned above can be used to detect and/or identify MRSA, whether said diagnostic kits, primers and probes are used forin vitroorin situapplications. The said samples may include but are not limited to: any clinical sample, any environmental sample, any microbial culture, any microbial colony, any tissue, and any cell line. 

[0032] It is also an object of the present invention that said diagnostic kits, primers and probes 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 cuture medium, any cellular stain, any culture on specific cell lines, and any infectivity assay on animals. 

[0033] In the methods and kits described herein below, the oligonucleotide probes and amplification primers have been derived from larger sequences (i.e. DNA fragments of at least 100 base pairs). All DNA sequences have been obtained either from our proprietary sequences or from public databases (Tables 5, 6, 7, 8 and 9). 

[0034] It is clear to the individual skilled in the art that oligonucleotide sequences other than those described in the present invention and which are appropriate for detection and/or identification of MRSA may also be derived from the proprietary fragment sequences or selected public database sequences. For example, the oligonucleotide primers or probes may be shorter but of a lenght of at least 10 nucleotides or longer than the ones chosen; they may also be selected anywhere else in the proprietary DNA fragments or in the sequences selected from public databases; they may also be variants of the same oligonucleotide. 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 said DNA fragment sequences for use in amplification methods other than PCR. Consequently, the core of this invention is the detection and/or identification of MRSA by targeting genomic DNA sequences which are used as a source of specific and ubiquitous oligonucleotide probes and/or amplification primers. Although the selection and evaluation of oligonucleotides suitable for diagnostic purposes require much effort, it is quite possible for the individual skilled in the art to derive, from the selected DNA fragments, oligonucleotides other than the ones listed in Tables 5, 6, 7, 8 and 9 which are suitable for diagnostic purposes. When a proprietary fragment or a public database sequence is selected for its specificity and ubiquity, it increases the probability that subsets thereof will also be specific and ubiquitous. 

[0035] The proprietary DNA fragments have been obtained as a repertory of sequences created by amplifying MRSA nucleic acids with new primers. These primers and the repertory of nucleic acids as well as the repertory of nucleotide sequences are further objects of this invention (Tables 4, 5, 6, 7, 8 and 9). 

[0036] Claims therefore are in accordance with the present invention. 

 SEQUENCES FOR DETECTION AND IDENTIFICATION OF MRSA 

[0037] In the description of this invention, the terms «nucleic acids» and «sequences» might be used interchangeably. However, «nucleic acids» are chemical entities while «sequences» are the pieces of information encoded by these «nucleic acids». Both nucleic acids and sequences are equivalently valuable sources of information for the matter pertaining to this invention. 

 Oligonucleotide primers and probes design and synthesis 

[0038] As part of the design rules, all oligonucleotides (probes for hybridization and primers for DNA amplification by PCR) were evaluated for their suitability for hybridization or PCR amplification by computer analysis using standard programs (i.e. the GCG Wisconsin package programs, the primer analysis softwareOligo™ 6 and MFOLD 3.0). The potential suitability of the PCR primer pairs was also 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 3' end ( Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C .). Oligonucleotide amplification primers were synthesized using an automated DNA synthesizer (Applied Biosystems). Molecular beacon designs were evaluated using criteria established by Krameret al.(http://www.molecular-beacons.org). 

[0039] 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) ( Nichols et al., 1994, Nature 369:492-493 ). Primers and probes may also consist of nucleotide analogs such as Locked Nucleic Acids (LNA) ( Koskinet al., 1998, Tetrahedron 54:3607-3630 ), and Peptide Nucleic Acids (PNA) ( Egholm et al., 1993, Nature 365:566-568 ). The primers or probes may be of any suitable length and may be selected anywhere within the DNA sequences from proprietary fragments, or from selected database sequences which are suitable for the detection of MRSA. 

[0040] Variants for a given target microbial gene are naturally occurring and are attributable to sequence variation within that gene during evolution ( Watsonet al., 1987, Molecular Biology of the Gene, 4th ed., The Benjamin/Cummings Publishing Company, Menlo Park, CA ; Lewin, 1989, Genes IV, John Wiley & Sons, New York, NY ). For example, different strains of the same microbial species may have a single or more nucleotide variation(s) at the oligonucleotide hybridization site. The person skilled in the art is well aware of 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. The detection of a variant sequence for a region between two PCR primers may be demonstrated by sequencing the amplification product. In order to show the presence of sequence variations at the primer hybridization site, one has to amplify a larger DNA target with PCR primers outside that hybridization site. Sequencing of this larger fragment will allow the detection of sequence variation at this primer hybridization site. A similar strategy may be applied to show 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 microbial DNA is under the scope of this invention. Variants of the selected primers or probes may also be used to amplify or hybridize to a variant target DNA. 

 DNA amplification 

[0041] For DNA amplification by the widely used PCR method, primer pairs were derived from our proprietary DNA fragments or from public database sequences. 

[0042] 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 exponentiallyin vitrothe 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 .). 

[0043] Briefly, the PCR protocols on a standard thermocycler (PTC-200 from MJ Research Inc., Watertown, MA) were as follows: Treated standardized bacterial suspensions or genomic DNA prepared from bacterial cultures or clinical specimens were amplified in a 20 µl PCR reaction mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 2.5 mM MgCI2, 0.4 µM of each primer, 200 µM of each of the four dNTPs (Pharmacia Biotech), 3.3 µg/µl bovine serum albumin (BSA) (Sigma-Aldrich CanadaLtd, Oakville, Ontario, Canada) and 0.5 unit ofTaqDNA polymerase (Promega Corp., Madison, WI) combined with the TaqStart™antibody (BD Biosciences, Palo Alto, CA). The TaqStart™ antibody, which is a neutralizing monoclonal antibody toTaq DNA polymerase, was added to all PCR reactions to enhance the specificity and the sensitivity of the amplifications ( Kellogg et al., 1994, Biotechniques 16:1134-1137 ). The treatment of bacterial cultures or of clinical specimens consists in a rapid protocol tolyse the microbial cells and eliminate or neutralize PCR inhibitors (described in co-pending application US 60/306,163 ). For amplification from purified genomic DNA, the samples were added directly to the PCR amplification mixture. An internal control, derived from sequences not found in the target MREJ sequences or in the human genome, was used to verify the efficiency of the PCR reaction and the absence of significant PCR inhibition. 

[0044] The number of cycles performed for the PCR assays varies according to the sensitivity level required. For example, the sensitivity level required for microbial detection directly from a clinical specimen is higher than for detection from a microbial culture. Consequently, more sensitive PCR assays having more thermal cycles are probably required for direct detection from clinical specimens. 

[0045] The person skilled in the art of nucleic acid amplification knows the existence of other rapid amplification procedures such as ligase chain reaction (LCR), reverse transcriptase PCR (RT-PCR), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), branched DNA (bDNA), cycling probe technology (CPT), solid phase amplification (SPA), rolling circle amplification technology (RCA), solid phase RCA, anchored SDA and nuclease dependent signal amplification (NDSA) ( Lee et al., 1997, Nucleic Acid Amplification Technologies: Application to Disease Diagnosis, Eaton Publishing, Boston, MA ; Persing et al., 1993, Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C .; Westin et al., 2000, Nat. Biotechnol. 18:199-204 ). The scope of this invention is not limited to the use of amplification by PCR, but rather includes the use of any nucleic acid amplification method or any other procedure which may be used to increase the sensitivity and/or the rapidity of nucleic acid-based diagnostic tests. The scope of the present invention also covers the use of any nucleic acids amplification and detection technology including real-time or post-amplification detection technologies, any amplification technology combined with detection, any hybridization nucleic acid chips or array technologies, any amplification chips or combination of amplification and hybridization chip technologies. Detection and identification by any nucleotide sequencing method is also under the scope of the present invention. 

[0046] Any oligonucleotide derived from the S.aureusMREJ DNA sequences and used with any nucleic acid amplification and/or hybridization technologies are also under the scope of this invention. 

 Evaluation of the MRSA detection method developed by Hiramatsuet al. 

[0047] According to Hiramatsuet al.( Ito et al., 1999, Antimicrob. Agents Chemother. 43:1449-1458 ; Katayama et al., 2000, Antimicrob. Agents Chemother. 44:1549-1555 ; Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-1336 , Ma et al., 2002, Antimicrob. Agents Chemother. 46:1147-1152 ), four types of SCCmecDNA are found among MRSA strains. They have found thatSCCmecDNAs are integrated at a specific site of the MSSA chromosome (namedorfX. They developed a MRSA-specific multiplex PCR assay including primers that can hybridize to the right extremity of SCCmectypes I, II and III (SEQ ID NOs.: 18, 19, 20, 21, 22, 23, 24 in US patent 6,156,507 corresponding to SEQ IDNOs.: 52, 53, 54, 55, 56, 57, 58, respectively, in the present invention) as well as primers specific to the S.aureuschromosome to the right of the SCCmecintegration site (SEQ ID NO.: 25, 28, 27, 26, 29 in US patent 6,156,507 corresponding to SEQ ID NOs.: 59, 60, 61, 62, 63, respectively, in the present invention) (Table 1 and Figure 1 ). The set of primers described by Hiramatsuet al.as being the optimal primer combination (SEQ ID NOs.: 22, 24 and 28 in US patent 6,156,507 corresponding to SEQ ID NOs.: 56, 58 and 60 in the present invention) was used in the present invention to test by PCR a variety of MRSA, MSSA, methicillin-resistant CNS (MRCNS) and methicillin-sensitive CNS (MSCNS) strains (Table 2). A PCR assay performed using a standard thermocycler (PTC-200 from MJ Research Inc.) was used to test the ubiquity, the specificity and the sensitivity of these primers using the following protocol: one µl of a treated standardized bacterial suspension or of a genomic DNA preparation purified from bacteria were amplified in a 20 µl PCR reaction mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgC12, 0.4 µM of each of the SCCmec- andS. aureuschromosome-specific primers (SEQ ID NOs.: 22, 24 and 28 in US patent 6,156,507 corresponding to SEQ IDNOs.: 56, 58 and 60 in the present invention), 200 µM of each of the four dNTPs (Pharmacia Biotech), 3.3 µg/µl BSA (Sigma), and 0.5 UTaqpolymerase (Promega) coupled withTaqStart™ Antibody (BD Biosciences). 

[0048] PCR reactions were then subjected to thermal cycling 3 min at 94°C followed by 40 cycles of 60 seconds at 95°C for the denaturation step, 60 seconds at 55°C for the annealing step, and 60 seconds at 72°C for the extension step, then followed by a terminal extension of 7 minutes at 72°C using a standardthermocycler (PTC-200 from MJ Research Inc.). Detection of the PCR products was made by electrophoresis in agarose gels (2 %) containing 0.25 µg/ml ofethidium bromide. Twenty of the 39 MRSA strains tested were not amplified with the PCR assay developed by Hiramatsuet al.(Example 1, Tables 2 and 3). 

[0049] With a view of establishing a rapid diagnostic test for MRSAs, the present inventors developed new sets of primers specific to the right extremity ofSCCmec types I and II (SEQ ID NOs.: 66, 100 and 101) (Annex 1), SCCmec type II (SEQ ID NOs.: 97 and 99), SCCmec type III (SEQ ID NOs.: 67, 98 and 102) and in the S. aureus chromosome to the right of the SCCmec integration site (SEQ ID NOs.: 64, 70, 71, 72, 73, 74, 75 and 76) (Table 5). These primers, amplifying short amplicons (171 to 278 bp), are compatible for use in rapid PCR assays (Table 7). The design of these primers was based on analysis of multiple sequence alignments of orfX and SCCmec sequences described by Hiramatsu et al. (US patent 6,156,507 ) or available from GenBank (Table 10, Annex I). These different sets of primers were used to test by PCR a variety of MRSA, MSSA, MRCNS and MSCNS strains. Several amplification primers were developed to detect all three SCCmec types (SEQ ID NOs.: 97 and 99 for SCCmec type II, SEQ ID NOs.: 66, 100 and 101 for SCCmec types I and II and SEQ ID NOs.: 67, 98 and 102 for SCCmec type III). Primers were chosen according to their specificity for MRSA strains, their analytical sensitivity in PCR and the length of the PCR product. A set of two primers was chosen for the SCCmec right extremity region (SEQ ID NO.: 66 specific to SCCmec types I and II; SEQ ID NO.: 67 specific to SCCmec type III). Of the 8 different primers designed to anneal on the S. aureus chromosome to the right of the SCCmec integration site (targeting orfX gene) (SEQ ID NOs.: 64, 70, 71, 72, 73, 74, 75 and 76), only one (SEQ ID.: 64) was found to be specific for MRSA based on testing with a variety of MRSA, MSSA, MRCNS and MSCNS strains (Table 12). Consequently, a PCR assay using the optimal set of primers (SEQ ID NOs.: 64, 66 and 67) which could amplify specifically MRSA strains containing SCCmec types I, II and III was developed ( Figure 2 , Annex I). While the PCR assay developed with this novel set of primers was highly sensitive (i.e allowed the detection of 2 to 5 copies of genome for all three SCCmec types) (Table 11), it had the same shortcomings (i.e. lack of ubiquity) of the test developed by Hiramatsu et al. The 20 MRSA strains which were not amplified by the Hiramatsu et al. primers were also not detected by the set of primers comprising SEQ ID NOs.: 64, 66 and 67 (Tables 3 and 12). Clearly, diagnostic tools for achieving at least 50% ubiquity amongst the tested strains are needed. 

[0050] With a view to establish a more ubiquitous (i.e. ability to detect all or most MRSA strains) detection and identification method for MRSA, we determined the sequence of the MREJ present in these 20 MRSA strains which were not amplified. This research has led to the discovery and identification of seven novel distinct MREJ target sequences which can be used for diagnostic purposes. These seven new MREJ sequences could not have been predicted nor detected with the system described in US patent 6,156,507 byHiramatsu et al.Namely, the present invention represents an improved method for the detection and identification of MRSA because it provides a more ubiquitous diagnostic method which allows for the detection of all major epidemic MRSA clones from around the world. 

 Sequencing of MREJ nucleotide sequences from MRSA strains not amplifiable with primers specific to SCCmec types I, II and III 

[0051] Since DNA from twenty MRSA strains were not amplified with the set of primers developed by Hiramatsuet al.(SEQ ID NOs.: 22, 24 and 28 in US patent 6,156,507 corresponding to SEQ IDNOs.: 56, 58 and 60 in the present invention) (Tables 2 and 3) nor with the set of primers developed in the present invention based on the same threeSCCmectypes (I, II and III) sequences (SEQ ID NOs.: 64, 66 and 67) (Table 12), the nucleotide sequence of the MREJ was determined for sixteen of these twenty MRSA strains. 

[0052] Transposase of IS431is often associated with the insertion of resistance genes within themeclocus. The gene encoding this transposase has been described frequently in one or more copies within the right segment of SCCmec( Oliveira et al., 2000, Antimicrob. Agents Chemother. 44:1906-1910 ; Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-36 ). Therefore, in a first attempt to sequence the novel MREJ for 16 of the 20 MRSA strains described in Table 3, a primer was designed in the sequence of the gene coding for the transposase of IS431 (SEQ ID NO.: 68) and combined with anorfXspecific primer to the right of theSCCmecintegration site (SEQ ID NO.: 70) (Tables 5 and 8). The strategy used to select these primers is illustrated in Figure 3 . 

[0053] The MREJ fragments to be sequenced were amplified using the following amplification protocol: one µL of treated cell suspension (or of a purifiedgenomic DNA preparation) was transferred directly into 4 tubes containing 39 µL of a PCR reaction mixture. Each PCR reaction contained 50mM KC1, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCI2, 1µM of each of the 2 primers (SEQ ID NOs.: 68 and 70), 200 µM of each of the four dNTPs, 3.3 µg/µl of BSA (Sigma-Aldrich Canada Ltd) and 0.5 unit ofTaqDNA polymerase (Promega) coupled with theTaqStart™ Antibody (BD Bisociences). PCR reactions were submitted to cycling using a standard thermocycler (PTC-200 from MJ Research Inc.) as follows: 3 min at 94 °C followed by 40 cycles of 5 sec at 95 °C for the denaturation step, 30 sec at 55 °C for the annealing step and 2 min at 72 °C for the extension step. 

[0054] Subsequently, the four PCR-amplified mixtures were pooled and 10 µL of the mixture were resolved by electrophoresis in a 1.2% agarose gel containing 0.25µg/mL of ethidium bromide. The amplicons were then visualized with an Alpha-Imager (Alpha Innotech Corporation, San Leandro, CA) by exposing to UV light at 254 nm. Amplicon size was estimated by comparison with a 1 kb molecular weight ladder (Life Technologies, Burlington, Ontario, Canada). The remaining PCR-amplified mixture (150 µL, total) was also resolved by electrophoresis in a 1.2% agarose gel. The amplicons were then visualized by staining with methylene blue ( Flores et al., 1992, Biotechniques, 13:203-205 ). Amplicon size was once again estimated by comparison with a 1 kb molecular weight ladder. Of the sixteen strains selected from the twenty described in Table 3, six were amplified using SEQ ID NOs.: 68 and 70 as primers (CCRI-178, CCRI-8895, CCRI-8903, CCRI-1324, CCRI-1331 and CCRI-9504). For these six MRSA strains, an amplification product of 1.2 kb was obtained. Theband corresponding to this specific amplification product was excised from theagarose gel and purified using the QIAquick™ gel extraction kit (QIAGEN inc., Chatsworth, CA). The gel-purified DNA fragment was then used directly in the sequencing protocol. Both strands of the MREJ amplification products were sequenced by the dideoxynucleotide chain termination sequencing method by using an Applied Biosystems automated DNA sequencer (model 377) with their Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA). The sequencing reactions were performed by using the same primers (SEQ ID NOs.: 68 and 70) and 10 ng/100 bp per reaction of the gel-purified amplicons. Sequencing of MREJ from the six MRSA strains (CCRI-178, CCRI-8895, CCRI-8903, CCRI-1324, CCRI-1331 and CCRI-9504) described in Table 3 yielded SEQ ID NOs.: 42, 43, 44, 45, 46 and 51, respectively (Table 4). 

[0055] In order to ensure that the determined sequence did not contain errors attributable to the sequencing of PCR artefacts, we have sequenced two preparations of the gel-purified MREJ amplification products originating from two independent PCR amplifications. For most target fragments, the sequences determined for both amplicon preparations were identical. Furthermore, the sequences of both strands were 100% complementary thereby confirming the high accuracy of the determined sequence. The MREJ sequences determined using the above strategy are described in the Sequence Listing and in Table 4. 

[0056] In order to sequence MREJ in strains for which no amplicon had been obtained using the strategy including primers specific to thetransposase gene of IS431 andorfX,another strategy using primers targetingmecAandorfXsequences was used to amplify longer genomic fragments. A new PCR primer targetingmecA(SEQ ID NO.: 69) (Table 8) to be used in combination with the same primer in theorfXsequence (SEQ ID NO.: 70). The strategy used to select these primers is illustrated in Figure 3 . 

[0057] The following amplification protocol was used: Purifiedgenomic DNA (300 ng) was transferred to a final volume of 50 µl of a PCR reaction mixture. Each PCR reaction contained 1X Herculase buffer (Stratagene, La Jolla, CA), 0.8 µM of each of the 2 primers (SEQ ID NOs.: 69 and 70), 0.56 mM of each of the four dNTPs and 5 units ofHerculase(Stratagene). PCR reactions were subjected to cycling using a standard thermal cycler (PTC-200 from MJ Research Inc.) as follows: 2 min at 92 °C followed by 35 or 40 cycles of 10 sec at 92 °C for thedenaturation step, 30 sec at 55 °C for the annealing step and 30 min at 68 °C for the extension step. 

[0058] Subsequently, 10 µL of the PCR-amplified mixture were resolved by electrophoresis in a 0.7% agarose gel containing 0.25 µg/mL ofethidium bromide. The amplicons were then visualized as described above. Amplicon size was estimated by comparison with a 1 kb molecular weight ladder (Life Technologies). A reamplification reaction was then performed in 2 to 5 tubes using the same protocol with 3 µl of the first PCR reaction used as test sample for the second amplification. The PCR-reamplified mixtures were pooled and also resolved by electrophoresis in a 0.7% agarose gel. The amplicons were then visualized by staining with methylene blue as described above. An amplification product of approximately 12 kb was obtained using this amplification strategy for all strains tested. The band corresponding to the specific amplification product was excised from the agarose gel and purified as described above. The gel-purified DNA fragment was then used directly in the sequencing protocol as described above. The sequencing reactions were performed by using the same amplification primers (SEQ ID NOs.: 69 and 70) and 425-495 ng of the gel-purified amplicons per reaction. Subsequently, internal sequencing primers (SEQ IDNOs.: 65, 77 and 96) (Table 8) were used to obtain sequence data on both strands for a larger portion of the amplicon. Five of the 20 MRSA strains (CCRI-1331, CCRI-1263, CCRI-1377, CCRI-1311 and CCRI-2025) described in Table 3 were sequenced using this strategy, yielding SEQ ID NOs.: 46, 47, 48, 49 and 50, respectively (Table 4). Sequence withinmecAgene was also obtained from the generated amplicons yielding SEQ ID NOs: 27, 28, 29, 30 and 31 from strains CCRI-2025, CCRI-1263, CCRI-1311, CCRI-1331 and CCRI-1377, respectively (Table 4). Longer sequences within themecAgene and from downstream regions were also obtained for strains CCRI-2025, CCRI-1331, and CCRI-1377 as described below. 

[0059] In order to obtain longer sequences of theorfXgene, two other strategies using primers targetingmecAandorfXsequences (at the start codon) was used to amplify longer chromosome fragments. A new PCR primer was designedin orfX(SEQ ID NO.: 132) to be used in combination with the same primer in themecAgene (SEQ ID NO.: 69). The strategy used to select these primers is illustrated in Figure 3 . Eight S.aureusstrains were amplified using primers SEQ ID NOs.: 69 and 132 (CCRI-9860, CCRI-9208, CCRI-9504, CCRI-1331, CCRI-9583, CCRI-9681, CCRI-2025 and CCRI-1377). The strategy used to select these primers is illustrated in Figure 3 . 

[0060] The following amplification protocol was used: Purified genomic DNA (350 to 500 ng) was transferred to a 50 µl PCR reaction mixture. Each PCR reaction contained 1XHerculase buffer (Stratagene), 0.8 µM of each of the set of 2 primers (SEQ ID NOs.: 69 and 132), 0.56 mM of each of the four dNTPs and 7.5 units ofHerculase(Stratagene) with 1 mM MgCl2. PCR reactions were subjected to thermocycling as described above. 

[0061] Subsequently, 5 µL of the PCR-amplified mixture were resolved by electrophoresis in a 0.8% agarose gel containing 0.25µg/mL ofethidium bromide. The amplicons were then visualized as described above. For oneS.aureusstrain (CCRI-9583), a reamplification was then performed by using primers SEQ ID NOs.: 96 and 158 ( Figure 3 ) in 4 tubes, using the same PCR protocol, with 2 µl of the first PCR reaction as test sample for the second amplification. The PCR-reamplified mixtures were pooled and also resolved by electrophoresis in a 0.8% agarose gel. The amplicons were then visualized by staining withmethylene blue as described above. A band of approximately 12 to 20 kb was obtained using this amplification strategy depending on the strains tested. Theband corresponding to the specific amplification product was excised from the agarose gel and purified using the QIAquick™ gel extraction kit or QIAEX II gel extraction kit (QIAGEN Inc.). Two strains, CCRI-9583 and CCRI-9589, were also amplified with primers SEQ ID NOs.: 132 and 150, generating an amplification product of 1.5 kb. Long amplicons (12-20 kb) were sequenced using 0.6 to 1 µg per reaction, while short amplicons (1.5 kb) were sequenced using 150 ng per reaction. Sequencing reactions were performed using different sets of primers for eachS.aureusstrain: 1) SEQ ID NOs.: 68, 70, 132, 145, 146, 147, 156, 157 and 158 for strain CCRI-9504; 2) SEQ ID NOs.: 70, 132, 154 and 155 for strain CCRI-2025; 3) SEQ ID NOs.: 70, 132, 148, 149, 158 and 159 for strain CCRI-9681; 4) SEQ IDNOs..: 70, 132, 187, and 188 for strain CCRI-9860; 5) SEQ IDNOs: 70, 132, 150 and 159 for strain CCRI-9589, 6) SEQ ID NOs.: 114, 123, 132, 150 and 158 for strain CCRI-9583; 7) SEQ ID NOs: 70, 132, 154 and 155 for strain CCRI-1377, 8) SEQ ID NOs.: 70, 132, 158 and 159 for strain CCRI-9208; 9) SEQ ID NOs: 68, 70, 132, 145, 146, 147 and 158 for strain CCRI-1331; and 10) SEQ IDNOs.: 126 and 127 for strain CCRI-9770. 

[0062] In one strain (CCRI-9770), theorfXandorfSA0022 genes were shown to be totally or partially deleted based on amplification using primers specific to these genes (SEQ ID NOs: 132 and 159 and SEQ ID NOs.: 128 and 129, respectively) (Table 8). Subsequently, a new PCR primer was designed inorfSA0021 (SEQ ID NO.: 126) to be used in combination with the same primer in themecAgene (SEQ ID NO.: 69). An amplification product of 4.5 kb was obtained with this primer set. Amplification, purification of amplicons and sequencing of amplicons were performed as described above. 

[0063] To obtain the sequence of the SSCmecregion containingmecAfor ten of the 20 MRSA strains described in Table 3 (CCRI-9504, CCRI-2025, CCRI-9208, CCRI-1331, CCRI-9681, CCRI-9860, CCRI-9770, CCRI-9589, CCRI-9583 and CCRI-1377), the primer described above designedin mecA(SEQ ID NO.: 69) was used in combination with a primer designed in the downstream regionofmecA(SEQ ID NO.: 118) (Table 8). An amplification product of 2 kb was obtained for all the strains tested. For one strain, CCRI-9583, a re-amplification with primers SEQ ID NOs.: 96 and 118 was performed with the amplicon generated with primers SEQ ID NOs.: 69 and 132 described above. The application, re-amplification, purification of amplicons and sequencing reactions were performed as described above. Sequencing reactions were performed with amplicons generated with SEQ ID NOs.: 69 and 132 described above or SEQ ID NOs.: 69 and 118. Different sets of sequencing primers were used for eachS aureusstrain: 1) SEQ ID NOs.: 69, 96, 117, 118, 120, 151, 152 for strains CCRI-9504, CCRI-2025, CCRI-1331, CCRI-9770 and CCRI-1377; 2) SEQ ID NOs.: 69, 96, 118 and 120 for strains CCRI-9208, CCRI-9681 and CCRI-9589; 3) SEQ ID NOs.: 69, 96, 117, 118, 120 and 152 for strain CCRI-9860; and 4) SEQ ID NOs.: 96, 117, 118, 119, 120, 151 and 152 for strain CCRI-9583. 

[0064] The sequences obtained for 16 of the 20 strains non-amplifiable by theHiramatsu assay (Table 4) were then compared to the sequences available from public databases. In all cases, portions of the sequence had an identity close to 100% to publicly available sequences fororfX(SEQ ID NOs.: 42-51, 165-168 and 171) ormecAand downstream region (SEQ ID NOs.: 27-31, 189-193, 195, 197-199 and 225). However, while theorfXportion of the fragments (SEQ ID NOs.: 42-51, 165-168 and 171) shared nearly 100% identity with theorfXgene of MSSA strain NCTC 8325 described by Hiramatsuet al.(SEQ ID NO.: 3), the DNA sequence within the right extremityofSCCmecitself was shown to be very different from those of types I, II, III and IV described by Hiramatsuet al.(Table 13, Figure 4 ). Six different novel sequence types were obtained. 

[0065] It should be notedthat Hiramatsu et al.demonstrated that SCCmectype I could be associated with MREP type i, SCCmectypes II and IV are associated with MREP type ii, and SCCmectype III is associated with MREP type iii. Our MREJ sequencing data from various MRSA strains led to the discovery of 6 novel MREP types designated types iv, v vi, vii, viii, and ix. The MREJ comprising distinct MREP types were named according to the MREP numbering scheme. Hence, MREP type i is comprised within MREJ type i, MREP type ii is comprised within MREJ type ii and so on up to MREP type ix. 

[0066] The sequences within the right extremity of SCCmecobtained from strains CCRI-178, CCRI-8895, CCRI-8903, CCRI-1324, CCRI-1331 and CCRI-9504 (SEQ ID NOs.: 42, 43, 44, 45, 46 and 51) were nearly identical to each other and exhibited nearly 100% identity with IS431(GenBank accession numbers AF422691, AB037671, AF411934). However, our sequence data revealed for the first time the location of this IS431sequence at the right extremity ofSCCmecadjacent to the integration site. Therefore, as the sequences at the right extremity ofSCCmecfrom these 6 MRSA strains were different from thoseofSCCmectype I from strain NCTC 10442, SCCmectype II from strain N315, SCCmectype III from strain 85/2082 and SCCmectype IV from strains CA05 and 8/6-3P described by Hiramatsuet al.( Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-1336 ; Ma et al., 2002, Antimicrob. Agents Chemother. 46:1147-1152 ), these new sequences were designated as MREP type iv (SEQ ID NOs.: 42-46 and 51). A BLAST search with the SCCmecportion of MREP type iv sequences produced significant alignments with sequences coding for portions of a variety of known transposases. For example, when compared to Genbank accession no. AB037671, MREP type iv from SEQ ID NO. 51 shared 98% identity with the putative transposase of IS431and its downstream region; two gaps of 7 nucleotides each were also present in the alignment. 

[0067] Sequences obtained from strains CCRI-1263, CCRI-1377, CCRI-1311 and CCRI-2025 (SEQ ID NOs.: 47-50) were nearly identical to each other and different from all three SCCmectypes and MREP type iv and, consequently, were designated as MREP type v. When compared with Genbank sequences using BLAST, MREP type v sequences did not share any significant homology with any published sequence, except for the first 28 nucleotides. That short stretch corresponded to the last 11 coding nucleotides oforfx,followed by the 17 nucleotides downstream, including the right inverted repeat (IR-R) of SCCmec.

[0068] Sequence obtained from strain CCRI-9208 was also different from all three SCCmectypes and MREP types iv and v and, consequently, was designated as MREP type vi (SEQ ID NO.: 171). Upon a BLAST search, MREP type vi was shown to be unique, exhibiting no significant homology to any published sequence. 

[0069] Sequences obtained from strains CCRI-9583 and CCRI-9589 were also different from all three SCCmectypes and MREP types iv to vi and were therefore designated as MREP type vii (SEQ ID NOs.: 165 and 166). Upon a BLAST search, MREP type vii was also shown to be unique, exhibiting no significant homology to any published sequence. 

[0070] Sequence obtained from strain CCRI-9860 was also different from all three SCCmectypes and MREP types iv to vii and was therefore designated as MREP type viii (SEQ ID NO.: 167). Sequence obtained from strain CCRI-9681 was also different from all three SCCmectypes and MREP types iv to viii and was therefore designated as MREP type ix (SEQ ID NO.: 168). BLAST searches with the SCCmecportion of MREP types viii and ix sequences yielded significant alignments, but only for the first ∼150 nucleotides of each MREP type. For example, the beginning of the MREP type viii sequence had 88% identity with a portion of Genbank accession no. AB063173, but no significant homology with any published sequence was found for the rest of the sequence. In the same manner, the first ∼150 nucleotides of MREP type ix had 97% identity with the same portion of AB063173, with the rest of the sequence being unique. The short homologous portion of MREP types viii and ix corresponds in AB063173 to the last 14 coding nucleotides oforfX,the IR-R of SCCmec,and a portion oforfCM009.Although sharing resemblances, MREP types viii and ix are very different from one another; as shown in Table 13, there is only 55.2% identity between both types for the first 500 nucleotides of the SCCmecportion. 

[0071] Finally, we did not obtain any sequence within SSCmecfrom strain CCRI-9770. However, as described in the section "Sequencing of MREJ nucleotide sequences from MRSA strains not amplifiable with primers specific to SCCmectypes I, II and III", this strain has apparently a partial or total deletion of theorfXandorfSA0022 genes in the chromosomal DNA to the right of theSCCmecintegration site and this would represent a new right extremity junction. We therefore designated this novel sequence as MREP type x (SEQ ID NO.: 172). Future sequencing should reveal whether this so called MREJ type x contains a novel MREP type x or if the lack of amplification is indeed caused by variation in the chromosomal part of the MREJ. 

[0072] The sequences of the first 500-nucleotide portion of the right extremity of all SCCmecobtained in the present invention were compared to thoseofSCCmectypes I, II and III using GCG programs Pileup and Gap. Table 13 depicts the identities at the nucleotide level betweenSCCmecright extremities of the six novel sequences with thoseofSCCmectypes I, II and III using the GCG program Gap. While SCCmectypes I and II showed nearly 79.2% identity (differing only by a 102 bp insertion presentinSCCmectype II) ( Figures 1 , 2 and 4 ), all other MREP types showed identities varying from 40.9 to 57.1%. This explains why the right extremities of the novel MREP types iv to ix disclosed in the present invention could not have been predicted nor detected with the system described by Hiramatsuet al.

[0073] Four strains (CCRI-1312, CCRI-1325, CCRI-9773 and CCRI-9774) described in Table 3 were not sequenced but rather characterized using PCR primers. Strains CCRI-1312 and CCRI-1325 were shown to contain MREP type v using specific amplification primers described in Examples 4, 5 and 6 while strains CCRI-9773 and CCRI-9774 were shown to contain R4REP type vii using specific amplification primers described in Example 7. 

[0074] To obtain the complete sequence of the SCCmecpresent in the MESA strains described in the present invention, primers targeting the S.aureuschromosome to the left (upstream of themecAgene) of the SCCmecintegration site were developed. Based on available public database sequences, 5 different primers were designed (SEQ ID NOs.: 85-89) (Table 9). These primers can be used in combination with S.aureuschromosome-specific primers in order to sequence the entire SCCmecor, alternatively, used in combination with amecA-specific primer (SEQ ID NO.: 81) in order to sequence the left extremity junction of SCCmec. We have also developed several primers specific to knownSCCmecsequences spread along the locus in order to obtain the complete sequence ofSCCmec(Table 9). These primers will allow to assign a SCCmectype to the MRSA strains described in the present invention. 

 Selection of amplification primers fromSCCmec /orlX sequences 

[0075] The MREJ sequences determined by the inventors or selected from public databases were used to select PCR primers for detection and identification of MRSA. The strategy used to select these PCR primers was based on the analysis of multiple sequence alignments of various MREJ sequences. 

[0076] Upon analysis of the six new MREP types iv to ixsequence data described above, primers specific to each new MREP type sequence (SEQ ID NOs.: 79, 80, 109, 112, 113, 115, 116 and 204) were designed ( Figure 2 , Table 5, Examples 3, 4, 5, 6, 7 and 8). Primers specific to MREP types iv, v and vii (SEQ IDNOs.: 79 , 80 and 112) were used in multiplex with the three primers to detectSCCmectypes I, II and III (SEQ ID NOs: 64, 66 and 67) and the primer specific to the Saureus orfX(SEQ ID NO. 64) (Examples 3, 4, 5, 6 and 7). Primers specific to MREP types vi, viii and ix (SEQ ID NOs.: 204, 115, 116 and 109) were also designed and tested against their specific target (Example 8). 

 Detection of amplification products 

[0077] Classically, the detection of PCR amplification products is performed by standard ethidium bromide-stained agarose gel electrophoresis as described above. It is however clear that other methods for the detection of specific amplification products, which may be faster and more practical for routine diagnosis, may be used. Examples of such methods are described in co-pending patent application WO01/23604 A2 . 

[0078] 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 our repertory and designed to specifically hybridize to DNA amplification products which are objects of the present invention. Alternatively,amplicons can be characterized by sequencing. See co-pending patent application WO01/23604 A2 for examples of detection and sequencing methods. 

[0079] In order to improve nucleic acid amplification efficiency, the composition of the reaction mixture may be modified ( 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 the use of various polymerases or the addition of nucleic acid amplification facilitators such asbetaine, BSA, sulfoxides, protein gp32, detergents, cations, tetramethylamonium chloride and others. 

[0080] In a preferred embodiment, real-time detection of PCR amplification was monitored using molecular beacon probes in a SmartCycler<®>apparatus (Cepheid, Sunnyvale, CA). A multiplex PCR assay containing primers specific to MREP types i to v andorfXof S.aureus(SEQ ID NOs.: 64, 66, 67, 79 and 80), a molecular beacon probe specific to theorfXsequence (SEQ ID NO. 84, see Annex II and Figure 2 ) and an internal control to monitor PCR inhibition was developed. The internal control contains sequences complementary to MREP type iv- andorfX-specific primers (SEQ ID NOs. 79 and and 64). The assay also contains a molecular beacon probe labeled with tetrachloro-6-carboxyfluorescein (TET) specific to sequence within DNA fragment generated during amplification of the internal control. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 3.45 mM MgCl2, 0.8 µm of each of the MREP-specific primers (SEQ ID NOs.: 66 and 67) andorfX-specific primer (SEQ ID NO.: 64), 0.4 µM of each of the MREP-specific primers (SEQ ID NOs.: 79 and 80), 80 copies of the internal control, 0.2 µm of the TET-labeled molecular beacon probe specific to the internal control, 0.2 µM of the molecular beacon probe (SEQ ID NO.: 84) labeled with 6-carboxyfluorescein (FAM), 330 µM of each of the four dNTPs (Pharmacia Biotech), 3.45 µg/µl of BSA (Sigma), and 0.875 UTaqpolymerase (Promega) coupled with TaqStart™Antibody (BD Biosciences). The PCR amplification on the Smart Cycler<®>was performed as follows: 3 min. at 95°C for initial denaturation, then forty-eight cycles of three steps consisting of 5 seconds at 95°C for the denaturation step, 15 seconds at 60°C for the annealing step and 15 seconds at 72°C for the extension step. Sensitivity tests performed by using purified genomic DNA from one MRSA strain of each MREP type (i to v) showed a detection limit of 2 to 10 genome copies (Example 5). None of the 26 MRCNS or 10 MSCNS tested were positive with this multiplex assay. The eight MRSA strains (CCRI-9208, CCRI-9770, CCRI-9681, CCRI-9860, CCRI-9583, CCRI-9773, CCRI-9774, CCRI-9589) which harbor the new MREP types vi, viii, ix and x sequences described in the present invention remained undetectable (Example 5). 

[0081] In a preferred embodiment, detection of MRSA using the real-time multiplex PCR assay on the Smart Cycler<®>apparatus (Cepheid, Sunnyvale, CA) directly from clinical specimens was evaluated. A total of 142 nasal swabs were collected during a MRSA hospital surveillance program at the Montreal General Hospital (Montreal, Quebec, Canada). The swab samples were tested at the Centre de Recherche en Infectiologie de l'Université Laval within 24 hours of collection. Upon receipt, the swabs were plated onto mannitol agar and then the nasal material from the same swab was prepared with a simple and rapid specimen preparation protocol described in co-pending patent application number US 60/306,163 . Classical identification of MRSA was performed by standard culture methods. 

[0082] The PCR assay detected 33 of the 34 samples positive for MRSA based on the culture method. As compared to culture, the PCR assay detected 8 additional MRSA positive specimens for a sensitivity of 97.1 % and a specificity of 92.6 % (Example 6). This multiplex PCR assay represents a rapid and powerful method for the specific detection of MRSA carriers directly from nasal specimens and can be used with any types of clinical specimens such as wounds, blood or blood culture, CSF, etc. 

[0083] In a preferred embodiement, a multiplex PCR assay containing primers specific to MREP types i, ii, iii, iv, v and vi and orfX of S. aureus (SEQ ID NOs.: 66, 67, 79, 80 and 112), and three molecular beacons probes specific toorfX sequence which allowed detection of the two sequence polymorphisms identified in this region of the orfX sequence was developed. Four of the strains which were not detected with the multiplex assay for the detection of MREP typesi to v were now detected with this multiplex assay while the four MRSA strains (CCRI-9208, CCRI-9770, CCRI-9681, CCRI-9860) which harbor the MREP types vi, viii, ix and x described in the present invention remained undetectable (Example 7). Primers soecific to MREP types vi, viii and ix (SEQ ID NOs.: 204, 115, 116 and 109) were also designed and were shown to detect their specific target, strains (Example 8). While the primers and probes derived from the teaching of Hiramatsuet al.,permitted the detection of only 48.7% (19 strains out of 39) of the MRSA strains of Table 2, the primers and probes derived from the present invention enable the detection of 97.4 % of the strains (38 strains out of 39) (seeexemples 7 and 8). Therefore it can be said that our assay has a ubiquity superior to 50% for the MRSA strains listed in Table 2. 

 Specificity, ubiquity and sensitivity tests for oligohucleotide primers and probes, 

[0084] The specificity of oligonucleotide primers and probes was tested by amplification of DNA or by hybridization with staphylococcal species. All of the staphylococcal species tested were likely to be pathogens associated with infections or potential contaminants which can be isolated from clinical specimens. Each target DNA could be released from microbial cells using standard chemical and/or physical treatments to lyse the cells(Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY ) or alternatively, genomic DNA purified with the GNOME™ DNA kit (Qbiogene, Carlsbad, CA) was used. Subsequently, the DNA was subjected to amplification with the set of primers. Specific primers or probes hybridized only to the target DNA. 

[0085] Oligonucleotides primers found to amplify specifically DNA from the target MRSA were subsequently tested for their ubiquity by amplification (i.e. ubiquitous primers amplified efficiently most or all isolates of MRSA). Finally, the analytical sensitivity of the PCR assays was determined by using 10-fold or 2-fold dilutions of purified genomic DNA from the targeted microorganisms. For most assays, sensitivity levels in the range of 2-10 genome copies were obtained. The specificity, ubiquity and analytical sensitivity of the PCR assays were tested either directly with bacterial cultures or with purified bacterial genomic DNA. 

[0086] Molecular beacon probes were tested using the Smart Cycler® platform as described above. A molecular beacon probe was considered specific only when it hybridized solely to DNA amplified from the MREJ ofS.aureus.Molecular beacon probes found to be specific were subsequently tested for their ubiquity (i.e. ubiquitous probes detected efficiently most or all isolates of the MRSA) by hybridization to bacterial DNAs from various MRSA strains. 

 Bacterial strains 

[0087] The reference strains used to build proprietarySCCmec-chromosome right extremity junction sequence data subrepeitories, as well as to test the amplification and hybridization assays, were obtained from (i) the American Type Culture Collection (ATCC), (ii) the Laboratoire de santé publique du Québec (LSPQ) (Ste-Anne de Bellevue, Québec, Canada), (iii) the Centers for Disease Control and Prevention (CDC) (Atlanta, GA), (iv) the Institut Pasteur (Paris, France), and V) the Harmony Collection (London, United Kingdom) (Table 14). Clinical isolates of MRSA, MSSA, MRCNS and MSCNS from various geographical areas were also used in this invention (Table 15). The identity of our MRSA strains was confirmed by phenotypic testing and reconfirmed by PCR analysis usings.aureus-specificprimers andmecA-specific primers (SEQ ID NOs.: 69 and 81) ( Martineau et al., 2000, Antimicrob. Agents Chemother. 44:231-238 ). 

 For sake of clarity, below is a list of the Examples, Tables, Figures and Annexes of this invention. 

 DESCRIPTION OF THE EXAMPLES 

[0088] Example 1: Primers developed by Hiramatsuet al.can only detect MRSA strains belonging to MREP types i, ii, and iii while missing prevalent novel MREP types. Example 2: Detection and identification of MRSA using primers specific to MREP types i, ii and iii sequences developed in the present invention. 

[0089] Example 3 : Development of a multiplex PCR assay on a standardthermocycler for detection and identification of MRSA based on MREP typesi, ii, iii, iv and v sequences. 

[0090] Example 4: Development of a real-time multiplex PCR assay on the Smart Cycler<®>for detection and identification of MRSA based on MREP typesi, ii, iii, iv and v sequences. 

[0091] Example 5: Development of a real-time multiplex PCR assay on the Smart Cycler<®>for detection and identification of MRSA based on MREP typesi, ii, iii, iv and v sequences and including an internal control. 

[0092] Example 6: Detection of MRSA using the real-time multiplex assay on the Smart Cyler<®>based on MREP types i, ii, iii, iv and v sequences for the detection of MRSA directly from clinical specimens. 

[0093] Example 7: Development of a real-time multiplex PCR assay on the Smart Cycler<®>for detection and identification of MRSA based on MREP typesi, ii, iii, iv, v, vi and vii sequences. 

[0094] Example 8: Developement of real-time PCR assays on the Smart Cycler® for detection and identification of MRSA based on MREP types vi, viii and ix. 

 DESCRIPTION OF THE TABLES 

[0095] 
Table 1 provides information about all PCR primers developed byHiramatsuet al.
in US patent 6,156,507 .
Table 2 is a compilation of results (ubiquity and specificity) for the detection of SCCmec-orfX right extremity junction using primers describedby Hiramatsu et al.
in US patent 6,156,507 on a standard thermocycler.
Table 3 is a list of MRSA strains not amplifiable using primers targeting types I, II and III of SCCmec -orfX right extremity junction sequences.
Table 4 is a list of novel sequences revealed in the present invention.
Table 5 provides information about all primers developed in the present invention.
Table 6 is a list of molecular beacon probes developed in the present invention.
Table 7 shows amplicon sizes of the different primer pairs described byHiramatsuet al. in US patent patent 6,156,507 or developed in the present invention.
Table 8 provides information about primers developed in the present invention to seequence the SCCmec -chromosome right extremity junction.
Table 9 provides information about primers developed in the present invention to obtain sequence of the complete SCCmec .
Table 10 is a list of the sequences available from public databases (GenBank, genome projects or US patent 6,156,507 ) used in the present invention to design primers and probes.
Table 11 gives analytical sensitivity of the PCR assay developed in the present invention using primers targeting types I, II and III of SCCme -orfX right extremity junction sequences and performed using a standard thermocycler.
Table 12 is a compilation of results (ubiquity and specificity) for the detection of MRSA using primers developed in the present invention which target types I, II and III of SCCmec-orfX right extremity junction sequences and performed using a standard thermocycler.
Table 13 shows a comparison of sequence identities between the first 500 nucleotides of SCCmec right extremities between 9 types of MREP.
Table 14 provides information about the reference strains of MRSA, MSSA, MRCNS and MSCNS used to validate the PCR assays developed in the present invention.
Table 15 provides information about the origin of clinical strains of MRSA, MSSA, MRCNS and MSCNS used to validate the PCR assays described in the present invention.
Table 16 depicts the analytical sensitivity of the PCR assay developed in the present invention using primers targeting 5 types of MREP sequences and performed on a standard thermocycler.
Table 17 is a compilation of results (ubiquity and specificity) for the PCR assay developed in the present invention using primers targeting 5 types of MREP sequences and performed on a standard thermocycler.
Table 18 depicts the analytical sensitivity of the PCR assay developed in the present invention using the Smart Cycler<®> platform for the detection of 5 types of MREP.
Table 19 is a compilation of results (ubiquity and specificity) for the PCR assay developed in the present invention using primers and a molecular beacon probe targeting 5 types of MREP sequences and performed on the Smart Cycler<®> platform.
Table 20 depicts the analytical sensitivity of the PCR assay developed in the present invention using the Smart Cycler<®> platform for the detection of 6 MREP types.
Table 21 is a compilation of results (ubiquity and specificity) for the PCR assay developed in the present invention using primers and a molecular beacon probe targeting 6 types of MREP sequences and performed on the Smart Cycler<®> platform.

 DESCRIPTION OF THE FIGURES 

[0096] 
Figure 1 is a diagram illustrating the position of the primers developed by Hiramatsu et al. (US patent 6,156,507 ) in the SCCmec -chromosome right extremity junction for detection and identification of MRSA.
Figure 2 is a diagram illustrating the position of the primers selected in the present invention in the SCCmec-orfX right extremity junction for detection and identification of MRSA.
Figure 3 is a diagram illustrating the position of the primers selected in the present invention to sequence new MREP types.
Figure 4 illustrates a sequence alignment of nine MREP types.

 FIGURE LEGENDS 

[0097] 
Figure 1 . Schematic organization of types I, II and III SCCmecorfX right extremity junctions and localization of the primers (SEQ ID NOs: 52-63) described by Hiramatsuet al. for the detection and identification of MRSA. Amplicon sizes are depicted in Table 7.
Figure 2 . Schematic organization of MREP types i, ii, iii, iv, v, vi, vii, viii and ix and localization of the primers and molecular beacon targeting all MREP types (SEQ ID NOs. 20, 64, 66, 67, 79, 80, 84, 112, 115, 116, 84, 163 and 164) which were developed in the present invention. Amplicon sizes are depicted in Table 7.
Figure 3 . Schematic organization of the SCCmec -chromosome right extremity junctions and localization of the primers (SEQ IDNOs. 65, 68, 69, 70, 77, 96, 118, 126, 132, 150 and 158) developed in the present invention for the sequencing of MREP types iv, v, vi, vii, viii, ix and x.
Figure 4 . Multiple sequence alignment of representatives of nine MREP types (represented by portions of SEQ IDNOs.: 1, 2, 104, 51, 50, 171, 165, 167 and 168 for types i, ii, iii, iv, v, vi, vii, viii and ix, respectively).

 DESCRIPTION OF THE ANNEXES 

[0098] The Annexes show the strategies used for the selection of primers and internal probes:
Annex I illustrates the strategy for the selection of primers fromSCCmec andorfX sequences specific for SCCmec types I and II.
Annex II illustrates the strategy for the selection of specific molecular beacon probes for the real-time detection ofSCCmec-orfX right extremity junctions.

[0099] As shown in these Annexes, the selected amplification primers may contain inosines and/or base ambiguities.Inosine is a nucleotide analog able to specifically bind to any of the four nucleotides A, C, G or T. Alternatively, degenerated oligonucleotides which consist of an oligonucleotide mix having two or more of the four nucleotides A, C, G or T at the site of mismatches were used. The inclusion of inosine and/or of degeneracies in the amplification primers allows mismatch tolerance thereby permitting the amplification of a wider array of target nucleotide sequences ( Dieffenbach and Dveksler, 1995, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, New York ). 

 EXAMPLES 

 EXAMPLE 1: 

 Primers developed by Hiramatsuet al. can only detect MRSA strains belonging to MREP types i, ii, and iii while missing prevalent novel MREP types. 

[0100] As shown in Figure 1 , Hiramatsuet al.have developed various primers that can specifically hybridize to the right extremities of types I, II and IIISCCmecDNAs. They combined these primers with primers specific to theS.aureuschromosome region located to the right of the SCCmecintegration site for the detection of MRSA. The primer set (SEQ ID NOs.: 22, 24 and 28 in US patent 6,156,507 corresponding to SEQ ID NOs.: 56, 58 and 60 in the present invention) was shown by Hiramatsuet al.to be the most specific and ubiquitous for detection of MRSA. This set of primers gives amplification products of 1.5 kb forSCCmectype I, 1.6 kb for SCCmectype II and 1.0 kb for SCCmectype III (Table 7). The ubiquity and specificity of this multiplex PCR assay was tested on 39 MRSA strains, 41 MSSA strains, 9 MRCNS strains and 11 MSCNS strains (Table 2). One µL of a treated standardized bacterial suspension or of a bacterial genomic DNA preparation. purified from bacteria were amplified in a 20 µl PCR reaction mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1 % Triton X-100, 2.5 mM MgCl2, 0.4 µM of each of the SCCmec-andorfX-specific primers (SEQ ID NOs.: 56, 58 and 60), 200 µM of each of the four dNTPs (Pharmacia Biotech), 3.3 µg/µl of BSA (Sigma), and 0.5 UTaqpolymerase (Promega) coupled withTaqStart™ Antibody (BD Biosciences). 

[0101] PCR reactions were then subjected to thermal cycling: 3 min at 94°C followed by 40 cycles of 60 seconds at 95°C for the denaturation step, 60 seconds at 55°C for the annealing step, and 60 seconds at 72°C for the extension step, then followed by a terminal extension of 7 minutes at 72°C using a standardthermocycler (PTC-200 from MJ Research Inc.). Detection of the PCR products was made by electrophoresis in agarose gels (2 %) containing 0.25 µg/ml of ethidium bromide. None of the MRCNS or MSCNS strains tested were detected with the set of primers detecting SCCmectypes I, II and III. Twenty of the 39 MRSA strains tested were not detected with this multiplex PCR assay (Tables 2 and 3). One of these undetected MRSA strains corresponds to the highly epidemic MRSA Portuguese clone (strain CCRI-9504; De Lencastre et al., 1994. Eur. J. Clin. Microbiol. Infect. Dis. 13:64-73 ) and another corresponds to the highly epidemic MRSA Canadian clone CMRSA1 (strain CCRI-9589; Simoret al.CCDR 1999, 25-12, june 15). These data demonstrate that the primer set developed byHiramatsu et al.(SEQ ID NOs.: 22, 24 and 28 in US patent 6,156,507 corresponding to SEQ ID NOs.: 56, 58 and 60 in the present invention) is not ubiquitous for the detection of MRSA and suggest that some MRSA strains have sequences at the SCCmec right extremity junction which are different from those identified by Hiramatsuet al.other types of SCCmecsequences or other sequences at the right extremity of SCCmec(MREP type) are found in MRSA. A limitation of this assay is the non-specific detection of 13 MSSA strains (Table 2). 

 EXAMPLE 2: 

[0102] Detection and identification of MRSA using primers specific to MREP types i, ii and iii sequences developed in the present invention. Based on analysis of multiple sequence alignments oforfXand SCCmecsequences described by Hiramatsuet al.or available from GenBank, a set of primers (SEQ ID NOs: 64, 66, 67) capable of amplifying short. segments of types I, II and IIIofSCCmec-orfxright extremity junctions from MRSA strains and discriminating from MRCNS (Annex I and Figure 2 ) were designed. The chosen set of primers gives amplification products of 176 bp for SCCmectype I, 278 pb for SCCmectype II and- 223 bp for SCCmectype III and allows rapid PCR amplification. These primers were used in multiplex PCR to test their ubiquity and specificity using 208 MRSA strains, 252 MSSA strains, 41 MRCNS strains and 21 MRCNS strains (Table 12). The PCR amplification and detection was performed as described in Example 1. PCR reactions were then subjected to thermal cycling (3 minutes at 94°C followed by 30 or 40 cycles of 1 second at 95°C for thedenaturation step and 30 seconds at 60°C for the annealing-extension step, and then followed by a terminal extension of 2 minutes at 72°C) using a standardthermocycler (PTC-200 from MJ Research Inc.). Detection of the PCR products was made as described in Example 1. 

[0103] None of the MRCNS or MSCNS strains tested were detected with this set of primers (Table 12). However, the twenty MRSA strains which were not detected with the primer set developed by Hiramatsuet al.(SEQ ID NOs: 56, 58 and 60) were also not detected with the primers developed in the present invention (Tables 3 and 12). These data also demonstrate that some MRSA strains have sequences at the SCCmec-chromosome right extremity junction which are different from those identified by Hiramatsuet al.Again, as observed with the Hiramatsu primers, 13 MSSA strains were also detected non-specifically (Table 12). The clinical significance of this finding remains to be established since these apparent MSSA strains could be the result of a recent deletion in themeclocus ( Deplano et al., 2000, J. Antimicrob. Chemotherapy, 46:617-619 ; Inglis et al., 1990, J. Gen. Microbiol,., 136:2231-2239 ; Inglis et al., 1993, J. Infect. Dis., 167:323-328 ; Lawrence et al. 1996, J. Hosp. Infect., 33:49-53 ; Wada et al., 1991, Biochem. Biophys. Res. Comm.,176:1319-1326 ). 

 EXAMPLE 3: 

[0104] Development of a multiplex PCR assay on a standard thermocycler for detection and identification of MRSA based on MREP types i, ii, iii, iv and v sequences. Upon analysis of two of the new MREP types iv and v sequence data described in the present invention, two new primers (SEQ ID NOs.: 79 and 80) were designed and used in multiplex with the three primers SEQ IDNOs.: 64, 66 and 67 described in Example 2. PCR amplification and detection of the PCR products was performed as described in Example 2. Sensitivity tests performed by using ten-fold or two-fold dilutions of purifiedgenomic DNA from various MRSA strains of each MREP type showed a detection limit of 5 to 10 genome copies (Table 16). Specificity tests were performed using 0,1 ng of purified genomic DNA or 1 µl of a standardized bacterial suspension. All MRCNS or MSCNS strains tested were negative with this multiplex assay (Table 17). Twelve of the 20 MRSA strains which were not detected with the multiplex PCR described in Examples 1 and 2 were now detected with this multiplex assay. Again, as observed with the Hiramatsu primers, 13 MSSA strains were also detected non-specifically (Table 12). The eight MRSA strains (CCRI-9208, CCRI-9583, CCRI-9773, CCRI-9774, CCRI-9589, CCRI-9860, CCRI-9681, CCRI-9770) and which harbor the new MREP types vi, vii, viii, ix and xsequences described in the present invention remained undetectable. 

 EXAMPLE 4: 

[0105] Development of a real-time multiplex PCR assay on the Smart Cycler<®>for detection and identification of MRSA based on MREP types i, ii, iii, iv and v sequences. The multiplex PCR assay described in Example 3 containing primers (SEQ ID NOs.: 64, 66, 67, 79 and 80) was adapted to the SmartCycler<®>platform (Cepheid). A molecular beacon probe specific to theorfXsequence was developed (SEQ ID NO. 84, see Annex II). Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1 % Triton X-100, 3.5 mM MgCl2, 0.4 µM of each of the SCCmec-andorfX-specific primers (SEQ ID NOs.: 64, 66, 67, 79 and 80), 0.2 µM of the FAM-labeled molecular beacon probe (SEQ ID NO.: 84), 200 µM of each of the four dNTPs, 3.3 µg/µl of BSA, and 0.5 UTaqpolymerase coupled withTaqStart™ Antibody. The PCR amplification on the Smart Cycler<®>was performed as follows: 3 min. at 94°C for initial denaturation, then forty-five cycles of three steps consisting of 5 seconds at 95°C for thedenaturation step, 15 seconds at 59°C for the annealing step and 10 seconds at 72°C for the extension step. Fluorescence detection was performed at the end of each annealing step. Sensitivity tests performed by using purified genomic DNA from several MRSA strains of each MREP type showed a detection limit of 2 to 10 genome copies (Table 18). None of the MRCNS or MSCNS were positive with this multiplex assay (Table 19). Again, as observed with the Hiramatsu primers, 13 MSSA strains were also detected non-specifically. Twelve of the twenty MRSA strains which were not detected with the multiplex PCR described in Examples 1 and 2 were detected by this multiplex assay. As described in Example 3, the eight MRSA strains which harbor the new MREP types vi, vii, viii, ix and x sequences described in the present invention remained undetectable. 

 EXAMPLE 5: 

[0106] Development of a real-time multiplex PCR assay on the Smart Cycler <®> for detection and identification of MRSA based on MREP types i, ii, iii, iv and v sequences including an internal control. The multiplex PCR assay described in Example 4 containing primers specific to MREP types to v andorfXofS. aureus(SEQ ID NOs.: 64, 66, 67, 79 and 80) and a molecular beacon probe specific to theorfXsequence (SEQ ID NO. 84, see Annex II) was optimized to include an internal control to monitor PCR inhibition. This internal control contains sequences complementary to MREP type iv- andorfXspecific primers (SEQ ID NOs. 79 and and 64). The assay also contains a TET-labeled molecular beacon probe specific to sequence within the amplicon generated by amplification of the internal control. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1 % Triton X-100, 3.45 mM MgCl2, 0.8 µM of each of the MREP-specific primers (SEQ ID NOs.: 66 and 67) andorfX-specific primer (SEQ ID NO.: 64), 0.4 µM of each of the MREP-specific primers (SEQ ID NOs.: 79 and 80), 80 copies of the internal control, 0.2 µM of the TET-labeled molecular beacon probe specific to the internal control, 0.2 µM of the FAM-labeled molecular beacon probe (SEQ ID NO.: 84), 330 µM of each of the four dNTPs (Pharmacia Biotech), 3.45 µg/µl of BSA (Sigma), and 0.875 UTaqpolymerase (Promega) coupled withTaqStart™ Antibody (BD Biosciences). The PCR amplification on the Smart Cycler<®>was performed as follows: 3 min. at 95°C for initial denaturation, then forty-eight cycles of three steps consisting of 5 seconds at 95°C for thedenaturation step, 15 seconds at 60°C for the annealing step and 15 seconds at 72°C for the extension step. Sensitivity tests performed by using purifiedgenomic DNA from one MRSA strain of each MREP type (i to v) showed a detection limit of 2 to 10 genome copies. None of the 26 MRCNS or 10 MSCNS were positive with this multiplex assay. Again, as observed with the Hiramatsu primers, 13 MSSA strains were also detected non-specifically. As described in Examples 3 and 4, the eight MRSA strains which harbor the new MREP types vi to x sequences described in the present invention remained undetectable. 

 EXAMPLE 6: 

[0107] Detection of MRSA using the real-time multiplex assay on the Smart Cycler® based on MREP types i, ii, iii, iv and v sequences directly from clinical specimens. The assay described in Example 5 was adapted for detection directly from clinical specimens. A total of 142 nasal swabs collected during a MRSA hospital surveillance program at the Montreal General Hospital (Montreal, Quebec, Canada) were tested. The swab samples were tested at the Centre de Recherche en Infectiologie de l'Université Laval within 24 hours of collection. Upon receipt, the swabs were plated onto mannitol agar and then the nasal material from the same swab was prepared with a simple and rapid specimen. preparation protocol described in co-pending patent application number US 60/306,163 . Classical identification of MRSA was performed by standard culture methods. 

[0108] The PCR assay described in Example 5 detected 33 of the 34 samples positive for MRSA based on the culture method. As compared to culture, the PCR assay detected 8 additional MRSA positive specimens for a sensitivity of 97.1 % and a specificity of 92.6 %. This multiplex PCR assay represents a rapid and powerful method for the specific detection of MRSA carriers directly from nasal specimens and can be used with any type of clinical specimens such as wounds, blood or blood culture, CSF, etc. 

 EXAMPLE 7 :

[0109] Development of a real-time multiplex PCR assay on the Smart Cycler<®>for detection and identification of MRSA based on MREP types i, ii, iii, iv, v and vii sequences. Upon analysis of the new MREP type vii sequence data described in the present invention (SEQ ID NOs.:165 and 166), two new primers (SEQ ID NOs.: 112 and 113) were designed and tested in multiplex with the three primers SEQ ID NOs.: 64, 66 and 67 described in Example 2. Primer SEQ ID NO.: 112 was selected for use in the multiplex based on its sensitivity. Three molecular beacon probes specific to theorfXsequence which allowed detection of two sequence polymorphisms identified in this region of theorfXsequence, based on analysis of SEQ ID NOs.: 173-186, were also used in the multiplex (SEQ IDNOs.: 84, 163 and 164). Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 3.45 mM MgCl2, 0.8 µM of each of the SCCmec-specific primers (SEQ ID NOs.: 66 and 67) andorfX-specific primer (SEQ ID NO.: 64), 0.4 µM of each of the SCCmec-specific primers (SEQ ID NOs.: 79 and 80), 0.2 µM of the FAM-labeled molecular beacon probe (SEQ ID NO.: 84), 330 µM of each of the four dNTPs (Pharmacia Biotech), 3.45 µg/µl of BSA (Sigma), and 0.875 U ofTαqpolymerase (Promega) coupled withTαqStart™Antibody (BD Biosciences). The PCR amplification on the Smart Cycler<®>was performed as follows: 3 min. at 95°C for initial denaturation, then forty-eight cycles of three steps consisting of 5 seconds at 95°C for the denaturation step, 15 seconds at 60°C for the annealing step and 15 seconds at 72°C for the extension step. The detection of fluorescence was done at the end of each annealing step. Sensitivity tests performed by using purified genomic DNA from several MRSA strains of each MREP type showed a detection limit of 2 genome copies (Table 20). None of the 26 MRCNS or 8 MSCNS were positive with this multiplex assay. Again, as observed with the Hiramatsu primers, 13 MSSA strains were also detected non-specifically (Table 21). Four of the strains which were not detected with the multiplex assay for the detection of MREP types i to v were now detected with this multiplex assay while the four MRSA strains (CCRI-9208, CCRI-9770, CCRI-9681, CCRI-9860) which harbor the MREP types vi, viii, ix and x described in the present invention remained undetectable. 

 EXAMPLE 8: 

[0110] Developement of real-time PCR assays on the Smart Cycler<®>for detection and identification of MRSA based on MREP types vi, viii, ix. Upon analysis of the new MREP types vi, viii and ix sequence data described in the present invention, one new primers specific to MREP type vi (SEQ ID NO.: 201), one primer specific to MREP type viii (SEQ ID NO.: 115), a primer specific to MREP type ix (SEQ ID NO.: 109) and a primer specific to both MREP types viii and ix (SEQ ID NO.: 116) were designed. Each PCR primer was used in combination with theorfX-specific primer (SEQ ID NO.: 64) and tested against its specific target strain. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 3.45 mM MgCl2, 0.4 µM of each of the SCCmec-andorfX-specific primers, 200 µM of each of the four dNTPs, 3.4 µg/µl of BSA, and 0.875 UTαqpolymerase coupled withTαqStart™ Antibody. The PCR amplification was performed as described en Example 7. Sensitivity tests performed by using genomic DNA purified from their respective MRSA target strains showed that the best primer pair combination was SEQ ID NOs.: 64 and 115 for the detection of MREP types viii and ix simultaneously. These newSCCmec-specific primers may be used in multiplex with primers specific to MREP types i, ii, ii, iv, v and vii (SEQ ID NOs.: 64, 66, 67, 79 and 80) described in previous examples to provide a more ubiquitous MRSA assay. 

[0111] In conclusion, we have improved the ubiquity of detection of MRSA strains. New MREJ types iv to x have been identified. Amongst strains representative of these new types, Hiramitsu's primers and/or probes succeeded in detecting less than 50% thereof. We have therefore amply passed the bar of at least 50% ubiquity, since our primers and probes were designed to detect 100% of the strains tested as representatives of MREJ types iv to ix. Therefore, although ubiquity depends on the pool of strains and representatives that are under analyse, we know now that close to 100% ubiquity is an attainable goal, when using the sequences of the right junctions (MREJ) to derive probes and primers dealing with polymorphism in this region. Depending on how many unknown types of MREJ exist, we have a margin of manoeuver going from 50% (higher than Hiramatsu's primers for the tested strains) to 100% if we sequence all the existing MREJs to derive properly the present diagnostic tools and methods, following the above teachings. 

[0112] This invention has been described herein above, and it is readily apparent that modifications can be made thereto without departing from the spirit of this invention. These modifications are under the scope of this invention, as defined in the appended claims .
Table 1. PCR amplification primers reported by Hiramatsu etal. in US patent 6,156,507 found in the sequence listing 
<tb>52MREP types i and ii48018
<tb>53MREP types i and ii75819
<tb>54MREP types i and ii92720
<tb>55MREP types i and ii115421
<tb>56MREP types i and ii175522
<tb>57MREP types i and ii230223
<tb>58MREP type iii295<c>24
<tb>59orfX166425
<tb>60orfSA0022<d>326728
<tb>61orfSA0022<d>358527
<tb>62orfX138926
<tb>63orfSA0022<d>295729
<tb><a>Position refers to nucleotide position of the 5' end of primer.
Numbering for SEQ ID NOs.: 52-57 refers to SEQ ID NO.: 2; numbering for SEQ ID NO.:
58 refers to SEQ ID NO.: 4; numbering for SEQ ID NOs.: 59-63 refers to SEQ ID NO.: 3.
<c>Primer is reverse-complement of target sequence.
<d>orfSA0022 refers to the open reading frame designation from GenBank accession number AP003129 (SEQ ID NO.: 231).
Table 2. Specificity and ubiquity tests performed on a standard thermocycler using the optimal set of primers described by Hiramatsu et al. (SEQ ID NOs 22, 24 and 28 in US patent 6,156,507 corresponding to SEQ ID NOs :56, 58 and 60 respectively, in the present invention) for the detection of MRSA 
<tb>MRSA - 39 strains19 (48.7)20 (51.2)
<tb>MSSA - 41 strains13 (31.7)28 (68.3)
<tb>MRCNS - 9 strains*0 (0%)9 (100%)
<tb>MSCNS - 11 strains*0 (0%)11 (100%)
<tb> * Details regarding CNS strains: 
MRCNS :S. caprae (1)
S. cohni cohnii(1)
S. epidermidis(1)
S. haemolyticus(2)
S. hominis (1)
S. sciuri (1)
S. simulans(1)
S. warneri(1)
MSCNS :S. cohni cohnii(1)
S. epidermidis (1)
S. equorum (1)
S. gallinarum(1)
S. haemolyticus(1)
S. lentus (1)
S. lugdunensis(1)
S. saccharolyticus(1)
S. saprophyticus(2)
S. xylosus (1)
Table 3. Origin of MESA strains not amplifiable using primers developed by Hiramatsu etal. (SEQ ID NOs.: 22, 24 and 28 in US patent 6,156,507 corresponding to SEQ ID NOs.: 56, 58 and 60, respectively, in the present invention) as well as primers developed in the present invention targeting MREP types i, ii and iii (SEQ ID NOs.: 64, 66 and 67) 
<tb>ATCC BAA-40CCRI-9504Portugal
<tb>ATCC 33592CCRI-178USA
<tb>R991282CCRI-2025Québec, Canada
<tb>4508CCRI-9208Québec, Canada
<tb>19121CCRI-8895Denmark
<tb>Z109CCRI-8903Denmark
<tb>45302CCRI-1263Ontario, Canada
<tb>R655CCRI-1324Québec, Canada
<tb>MA 50428CCRI-1311Québec, Canada
<tb>MA 50609CCRI-1312Québec, Canada
<tb>MA 51363CCRI-1331Québec, Canada
<tb>MA 51561CCRI-1325Québec, Canada
<tb>14A0116CCRI-9681Poland
<tb>23 (CCUG 41787)CCRI-9860Sweden
<tb>SE26-1CCRI-9770Ontario, Canada
<tb>SE1-1CCRI-9583Ontario, Canada
<tb>ID-61880<c>CCRI-9589Ontario, Canada
<tb>SE47-1CCRI-9773Ontario, Canada Ontario,
<tb>SE49-1CCRI-9774Ontario, Canada
<tb>39795-2CCRI-1377Québec, Canada
<tb><a>CCRI stands for "Collection of the Centre de Recherche en Infectiologie".
Portuguese clone.
<c>Canadian clone EMRSAI.
Table 4.Staphylococcus aureusMREJ nucleotide sequences revealed in the present invention 
<tb>27R991282CCRI-2025mecA
<tb>2845302CCRI-1263mecA
<tb>29MA 50428CCRI-1311mecA
<tb>30MA 51363CCRI-1331mecA
<tb>3139795-2CCRI-1377mecAand 1.5 kb of downstream region
<tb>42ATCC 33592CCRI-178MREP type iv
<tb>4319121CCRI-8895MREP type iv
<tb>44Z109CCRI-8903MREP type iv
<tb>45R655CCRI-1324MREP type iv
<tb>46MA 51363CCRI-1331MREP type iv
<tb>4745302CCRI-1263MREP type v
<tb>4839795-2CCRI-1377MREP type v
<tb>49MA 50428CCRI-1311MREP type v
<tb>50R991282CCRI-2025MREP type v
<tb>51ATCC BAA-40CCRI-9504MREP type iv
<tb>165SE1-1CCRI-9583MREP type vii
<tb>166ID-61880CCRI-9589MREP type vii
<tb>16723 (CCUG 41787)CCRI-9860MREP type viii
<tb>16814A016CCRI-9681MREP type ix
<tb>1714508CCRI-9208MREP type vi
<tb>172SE26-1CCRI-9770orfSA0021and 75 bp oforfSA0022
<tb>17326 (98/10618)CCRI-9864MREP type ii
<tb>17427 (98/26821)CCRI-9865MREP type ii
<tb>17528 (24344)CCRI-9866MREP type ii
<tb>17612 (62305)CCRI-9867MREP type ii
<tb>17722 (90/14719)CCRI-9868MREP type ii
<tb>17823 (98/14719)CCRI-9869MREP type ii
<tb>17932 (97S99)CCRI-9871MREP type ii
<tb>18033 (975100)CCRI-9872MREP type ii
<tb>18138 (825/96)CCRI-9873MREP type ii
<tb>18239 (842/96)CCRI-9874MREP type ii
<tb>18343 (N8-892/99)CCRI-9875MREP type ii
<tb>18446 (9805-0137)CCRI-9876MREP type iii
<tb>1851CCRI-9882MREP type ii
<tb>18629CCRI-9885MREP type ii
<tb>189SE1-1CCRI-9583mecAand 2.2 kb of downstream region, including IS431mec
<tb>190ATCC BAA-40CCRI-9504mecAand 1.5 kb of downstream region
<tb>1914508CCRI-9208mecAand 0.9 kb of downstream region
<tb>192ID-61880CCRI-9589mecAand 0.9 kb of downstream region
<tb>19314A016CCRI-9681mecAand 0.9 kb of downstream region
<tb>195SE26-1CCRI-9770mecAand 1.5 kb of downstream region, including IS431mec
<tb>197ATCC 43300CCRI-175MREP type ii
<tb>198R522CCRI-1262MREP type iii
<tb>19913370CCRI-8894MREP type i
<tb>219ATCC BAA-40CCRI-9504tetK
Table 4.Staphylococcus aureusMREJ nucleotide sequences revealed in the present invention (continued) 
<tb>
<tb>220MA 51363CCRI-1331mecA and 1.5 kb of downstream region
<tb>22139795-2CCRI-1377IS431mecand 0.6 kb of upstream region
<tb>222R991282CCRI-2025mecAand 1.5 kb of downstream region
<tb>223R991282CCRI-2025IS431mecand 0.6 kb of upstream region
<tb>22423 (CCUG 41787)CCRI-9860mecAand 1.5 kb of downstream region
<tb>22523 (CCUG 41787)CCRI-9860IS431mecand 0.6 kb of upstream region
<tb>23314A016CCRI-9681MREP type ix
<tb><a>CCRI stands for "Collection of the Centre de Recherche en Infectiologie".
orfSA0021 andorfSA0022 refer to the open reading frame designation from GenBank accession number AP003129 (SEQ ID NO.: 231).
Table 5. PCR primers developed in the present invention 
<tb>
<tb>64orfX17203
<tb>70orfX17963
<tb>71orfX17123
<tb>72orfX17493
<tb>73orfX17583
<tb>74orfX17943
<tb>75orfX17973
<tb>76orfX17983
<tb>66MREP types i and ii23272
<tb>100MREP types i and ii23232
<tb>101MREP types i and ii23142
<tb>97MREP type ii24342
<tb>99MREP type ii24342
<tb>67MREP type iii2074
<tb>98MREP type iii1474
<tb>102MREP type iii2514
<tb>79MREP type iv7443
<tb>80MREP type v5047
<tb>109MREP type ix652168
<tb>204MREP type vi642171
<tb>112MREP type vii503165
<tb>113MREP type vii551165
<tb>115MREP type viii514167
<tb>116MREP type viii601167
<tb><a>Position refers to nucleotide position of 5' end of primer.
Primer is reverse-complement of target sequence.
Table 6. Molecular beacon probes developed in the present invention 
<tb>32orfX86<a>
<tb>83orfX86<a>
<tb>84orfX34<a,b>
<tb>160orfX55<a,b>
<tb>161orfX34<a,b>
<tb>162orfX114<a>
<tb>163orfX34<a,b>
<tb>164orfX34<a,b>
<tb><a>Position refers to nucleotide position of the 5' end of the molecular beacon's loop on SEQ ID NO.: 3.
Sequence of molecular beacon's loop is reverse-complement of SEQ ID NO.: 3.
Table 7. Length of amplicons obtained with the different primer pairs which are objects of the present invention 
<tb>59/52orfX/MREP type i and ii2079 (type i) ; 2181 (type ii)
<tb>59/53orfX/MREP type i and ii1801 (type i) ; 1903 (type ii)
<tb>59/54orfX/MREP type i and ii1632 (type i) ; 1734 (type ii)
<tb>59/55orfX/MREP type i and ii1405 (type i) ; 1507 (type ii)
<tb>59/56orfX/MREP type i and ii804 (type i) ; 906 (type ii)
<tb>59/57orfX/MREP type i and ii257 (type i) ; 359 (type ii)
<tb>60/52orfSA0022/MREP type i and ii2794 (type i) ; 2896 (type ii)
<tb>60/53orfSA0022/MREP type i and ii2516 (type i) ; 2618 (type ii)
<tb>60/54orfSA0022/MREP type i and ii2347 (type i) ; 2449 (type ii)
<tb>60/55orfSA0022/MREP type i and ii2120 (type i) ; 2222 (type ii)
<tb>60/56orfSA0022/MREP type i and ii1519 (type i) ; 1621 (type ii)
<tb>60157orfSA0022/MREP type i and ii972 (type i) ; 1074 (type ii)
<tb>61/52orfSA0022/MREP type i and ii2476 (type i) ; 2578 (type ii)
<tb>61/53orfSA0022/MREP type i and ii2198 (type i) ; 2300 (type ii)
<tb>61/54orfSA0022/MREP type i and ii2029 (type i) ; 2131 (type ii)
<tb>61/55orfSA0022/MREP type i and ii1802 (type i) ; 1904 (type ii)
<tb>61/56orfSA0022/MREP type i and ii1201 (type i) ; 1303 (type ii)
<tb>61/57orfSA0022/MREP type i and ii654 (type i) ; 756(type ii)
<tb>62/52orfX/MREP type i and ii2354 (type i) ; 2456 (type ii)
<tb>62/53orfX/MREP type i and ii2076 (type i) ; 2178 (type ii)
<tb>62/54orfX/MREP type i and ii1907 (type i) ; 2009 (type ii)
<tb>62/55orfX/MREP type i and ii1680 (type i) ; 1782 (type ii)
<tb>62/56orfX/MREP type i and ii1079 (type i) ; 1181 (type ii)
<tb>62/57orfX/MREP type i and ii532 (type i) ; 634 (type ii)
<tb>63/52orfSA0022/MREP type i and ii3104 (type i) ; 3206 (type ii)
<tb>63/53orfSA0022/MREP type i and ii2826 (type i) ; 2928 (type ii)
<tb>63/54orfSA0022/MREP type i and ii2657 (type i) ; 2759 (type ii)
<tb>63/55orfSA0022/MREP type i and ii2430 (type i) ; 2532 (type ii)
<tb>63/56orfSA0022/MREP type i and ii1829 (type i) ; 1931 (type ii)
<tb>63/57orfSA0022/MREP type i and ii1282 (type i) ; 1384 (type ii)
<tb>59/58orfX/MREP type iii361
<tb>60/58orfSA0022/MREP type iii1076
<tb>61/58orfSA0022/MREP type iii758
<tb>62/58orfX/MREP type iii656
<tb>63/58orfSA0022/MREP type iii1386
<tb>70/66orfX/MREP type i and ii100 (type i) ; 202 (type ii)
<tb>70/67orfX/MREP type iii147 (type iii)
<tb>64/66<c>orfX/MREP type i and ii176 (type i);278 (type ii)
<tb>64/67<c>orfX/MREP type iii223
<tb>64/79<c>orfX/MREP type iv215
<tb>64/80<c>orfX/MREP type v196
<tb>64/97<c>orfX/MREP type ii171
<tb>64/98<c>orfX/MREP type iii163
<tb>64/99<c>orfX/MREP type ii171
<tb>64/100<c>orfX/MREP types i and ii180 (type i);282 (type ii)
<tb>64/101<c>orfX/MREP types i and ii189 (type i);291 (type ii)
<tb>64/102<c>orfX/MREP type iii263
<tb>64/109<c>orfX/MREP type ix369
<tb>64/204<c>orfX/MREP type vi348
<tb>64/112<c>orfX/MREP type vii214
<tb>64/113<c>orfX/MREP type vii263
<tb>64/115<c>orfX/MREP type viii227
<tb>64/116<c>orfX/MREP type viii318
<tb><a>Amplicon length is given in base pairs for MREP types amplified by the set of primers.
Set of primers described by Hiramatsu etal.in US patent 6,156,507.
<c>Set of primers developed in the present invention.
<d>orfSA0022 refers to the open reading frame designation from GenBank accession number AP003129 (SEQ ID NO.: 231).
Table 8. Other primers developed in the present invention 
<tb>77MREP type iv99343
<tb>65MREP type v63647
<tb>70orfX17963
<tb>68IS43162692
<tb>69mecA105978
<tb>96mecA194978
<tb>81mecA120678
<tb>114MREP type vii629165
<tb>117MREP type ii856194
<tb>118MREP type ii974194
<tb>119MREP type vii404189
<tb>120MREP type vii477189
<tb>123MREP type vii551165
<tb>124MREP type ii584170
<tb>125MREP type ii689170
<tb>126orfSA0021336231
<tb>127orfSA0021563231
<tb>128orfSA0022<d>2993231
<tb>129orfSA0022<d>3467231
<tb>132orfX3700231
<tb>145MREP type iv98851
<tb>146MREP type v138651
<tb>147MREP type iv89151
<tb>148MREP type ix664168
<tb>149MREP type ix849168
<tb>150MREP type vii1117165
<tb>151MREP type vii1473189
<tb>152IS431mec1592189
<tb>154MREP type v99650
<tb>155MREP type v93550
<tb>156tetkfrom plasmid pT1811169228
<tb>157tetkfrom plasmid pT181136228
<tb>158orfX27142
<tb>159orfX25392
<tb>187MREP type viii967167
<tb>188MREP type viii851167
<tb><a>Position refers to nucleotide position of the 5' end of primer.
Primer is reverse-complement of target sequence.
Table 9. Amplification and/or sequencing primers developed in the present invention 
<tb>85S. aureuschromosome19735
<tb>86S. aureuschromosome19837
<tb>87S. aureuschromosome19738
<tb>88S. aureuschromosome126539
<tb>89S. aureuschromosome18923
<tb>103orfX13863
<tb>105MREP type i23352
<tb>106MREP type ii24372
<tb>107MREP type iii1534
<tb>108MREP type iii1534
<tb>121MREP type vii1150165
<tb>122MREP type vii1241165
<tb>130orfX4029231
<tb>131region betweenorfSA0022 andorfSA0023<d>3588231
<tb>133merBfrom plasmid pI258262226
<tb>134merBfrom plasmid pI258539226
<tb>135merRfrom plasmid pI258564226
<tb>136merRfrom plasmid pI258444227
<tb>137merRfrom plasmid pI258529227
<tb>138merRfrom plasmid pI258530227
<tb>139rep from plasmid pUB110796230
<tb>140rep from plasmid pUB110761230
<tb>141rep from plasmid pUB110600230
<tb>142aadDfrom plasmid pUB1101320229
<tb>143aadDfrom plasmid pUB110759229
<tb>144aadDfrom plasmid pUB110646229
<tb>153MREP type vii1030165
<tb>200orfSA0022<d>871<c>231
<tb>201orfSA0022<d>1006231
<tb>202MREP type vi648171
<tb>203MREP type vi883171
<tb>205MREP type ix1180168
<tb>206MREP type ix1311233
<tb>207MREP type viii1337167
<tb>208MREP type viii1441167
<tb>209ccrA184232
<tb>210ccrA385232
<tb>211ccrA643232
<tb>212ccrA1282232
<tb>213ccrB1388232
<tb>214ccrB1601232
<tb>215ccrB2139232
<tb>216ccrB2199232
<tb>217ccrB2847232
<tb>218ccrB2946232
<tb><a>Position refers to nucleotide position of the 5' end of primer.
Primer is reverse-complement of target sequence.
<c>Primer contains two mismatches.
<d>orfSA0022 andorfSA0023 refer to the open reading frame designation from GenBank accession number AP003129 (SEQ ID NO.: 231).
Table 10. Origin of the nucleic acids and/or sequences available from public databases found in the sequence listing 
<tb>1NCTC 10442DatabaseAB033763SCCmectype I MREJ
<tb>2N315DatabaseD86934SCCmectype II MREJ
<tb>3NCTC 8325DatabaseAB014440MSSA chromosome
<tb>486/560DatabaseAB013471SCCmectype III MREJ
<tb>586/961DatabaseAB013472SCCmectype III MREJ
<tb>685/3907DatabaseAB013473SCCmectype III MREJ
<tb>786/2652DatabaseAB013474SCCmectype III MREJ
<tb>886/1340DatabaseAB013475SCCmectype III MREJ
<tb>986/1762DatabaseAB013476SCCmec type III MREJ
<tb>1086/2082DatabaseAB013477SCCmectype III MREJ
<tb>1185/2111DatabaseAB013478SCCmectype III MREJ
<tb>1285/5495DatabaseAB013479SCCmectype III MREJ
<tb>1385/1836DatabaseAB013480SCCmectype III MREJ
<tb>1485/2147DatabaseAB013481SCCmectype III MREJ
<tb>1585/3619DatabaseAB013482SCCmectype III MREJ
<tb>1685/3566DatabaseAB013483SCCmec type III MREJ
<tb>1785/2232DatabaseAB014402SCCmectype II MREJ
<tb>1885/2235DatabaseAB014403SCCmectype II MREJ
<tb>19MR108DatabaseAB014404SCCmectype II MREJ
<tb>2085/9302DatabaseAB014430SCCmec type I MREJ
<tb>2185/9580DatabaseAB014431SCCmectype I MREJ
<tb>2285/1940DatabaseAB014432SCCmectype I MREJ
<tb>2385/6219DatabaseAB014433SCCmectype I MREJ
<tb>2464/4176DatabaseAB014434SCCmectype I MREJ
<tb>2564/3846DatabaseAB014435SCC1mectype I MREJ
<tb>26HUC19DatabaseAF181950SCCmec type II MREJ
<tb>33G3US 6,156,507SEQ ID NO.: 15S. epidermidisSCCmectype II MREJ
<tb>34SH 518US 6,156,507SEQ ID NO.: 16S. haemolyticusSCCmec type II MREJ
<tb>35ATCC 25923US 6,156,507SEQ ID NO.: 9S.aureuschromosome
<tb>36STP23US 6,156,507SEQ ID NO.: 10S. aureuschromosome
<tb>37STP43US 6,156,507SEQ ID NO.: 12S. aureuschromosome
<tb>38STP53US 6,156,507SEQ ID NO.: 13S. aureuschromosome
<tb>39476Genome project<c>S. aureuschromosome
<tb>40252Genome project<c>SCCmectype II MREJ
<tb>41COLGenome project<d>SCCmectype I MREJ
<tb>78NCTC 8325DatabaseX52593mecA
<tb>82NCTC 10442DatabaseAB033763mecA
<tb>90N315DatabaseD86934mecA
<tb>9185/2082DatabaseAB037671mecA
<tb>92NCTC 10442DatabaseAB033763IS431
<tb>93N315DatabaseD86934IS431
<tb>94HUC19DatabaseAF181950IS431
<tb>95NCTC 8325DatabaseX53818IS431
<tb>10485/2082DatabaseAB037671SCCmectype III MREJ
<tb>226unknownDatabaseL29436merBon plasmid pI258
<tb>227unknownDatabaseL29436merRon plasmid pI258
<tb>228unknownDatabaseS67449tetKon plasmid pT181
<tb>229HUC19DatabaseAF181950aadDon plasmid pUB110
<tb>230HUC19DatabaseAF181950rep on plasmid pUB110
<tb>231N315DatabaseAP003129orfSA0021,orfSA0022,orfSA0023
<tb>23285/2082DatabaseAB037671ccrA/ccrB
<tb><a>MREJ refers to mec right extremity junction and includes sequences from SCCmec-right extremity and chromosomal DNA to the right of SCCmecintegration site.
Unless otherwise specified, all sequences were obtained from S. aureus strains.
<c>Sanger Institute genome project (http://www.sanger.ac.uk).
<d>TIGR genome project (http://www.tigr.org).
Table 11. Analytical sensitivity of the MRSA-specific PCR assay targeting MREP types i, ii and iii on a standard thermocycler using the set of primers developed in the present invention (SEQ ID NOs.: 64, 66 and 67) 
<tb>13370CCRI-8894 (I)5
<tb>ATCC 43300CCRI-175 (II)2
<tb>35290CCRI-1262 (III)2
<tb><a>CCRI stands for "Collection of the Centre de Recherche en Infectiologie".
Table 12. Specificity and ubiquity tests performed on a standard thermocycler using the set of primers targeting MREP types i, ii and iii developed in the present invention (SEQ ID NOs.: 64, 66 and 67) for the detection of MRSA 
<tb>MRSA - 208 strains188 (90.4)20 (9.6)
<tb>MSSA - 252 strains13 (5.2)239 (94.8)
<tb>MRCNS - 41 strains*042 (100)
<tb>MSCNS - 21 strains*021 (100)
<tb>* Details regarding CNS strains:
MRCNS:S. caprae(2)
S. cohni cohnii (3)
S. cohni urealyticum (4)
S. epidermidis(8)
S. haemolyticus (9)
S. hominis (4)
S. sciuri (4)
S. sciuri sciuri (1)
S. simulans (3)
S. warneri (3)
MSCHS:S. cohni cohnii(1)
S. epidermidis (3)
S. equorum (2)
S. felis (1)
S. gallinarum (1)
S. haemolyticus (1)
S. hominis (1)
S. lentus(1)
S. lugdunensis (1)
S. saccharolyticus (1)
S. saprophyticus (5)
S. simulans (1)
S. xylosus (1)
Table 13. Percentage of sequence identity for the first 500 nucleotides of SCCmecright extremities between all 9 types of MREPa,b 
<tb>i--79.242.842.841.244.444.642.342.1
<tb>ii43.947.544.741.745.052.057.1
<tb>iii46.844.542.945.042.845.2
<tb>iv45.841.444.348.041.3
<tb>v45.443.747.544.3
<tb>vi45.141.147.2
<tb>vii42.840.9
<tb>viii55.2
<tb>ix--
<tb><a>"First 500 nucleotides" refers to the 500 nucleotides within the SCCmecright extremity, starting from the integration site of SCCmec in theStaphylococcusaureus chromosome as shown on Figure 4 .
Sequences were extracted from SEQ ID NOs.: 1, 2, 104, 51, 50, 171, 165, 167, and 168 for types i to ix, respectively.
Table 14. Reference strains used to test Sensitivity and/or specificity and/or ubiquity of the MRSA-specific PCR assays targeting MREJ, sequences 
<tb>33591ATCC
<tb>33592ATCC
<tb>33593ATCC
<tb>BAA-38ATCC
<tb>BAA-39ATCC
<tb>BAA-40ATCC
<tb>BAA-41ATCC
<tb>BAA-42ATCC
<tb>BAA-43ATCC
<tb>BAA-44ATCC
<tb>F182CDC
<tb>23 (CCUG 41787)HARMONY Collection
<tb>ID-61880 (EMRSA1)LSPQ
<tb>MA 8628LSPQ
<tb>MA 50558LSPQ
<tb>MA 50428LSPQ
<tb>MA 50609LSPQ
<tb>MA 50884LSPQ
<tb>MA 50892LSPQ
<tb>MA 50934LSPQ
<tb>MA 51015LSPQ
<tb>MA 51056LSPQ
<tb>MRSA (n = 45)MA 51085LSPQ
<tb>MA 51172LSPQ
<tb>MA 51222LSPQ
<tb>MA 51363LSPQ
<tb>MA 51561LSPQ
<tb>MA 52034LSPQ
<tb>MA 52306LSPQ
<tb>MA 51520LSPQ
<tb>MA 51363LSPQ
<tb>98/10618HARMONY Collection
<tb>98/26821HARMONY Collection
<tb>24344HARMONY Collection
<tb>62305HARMONY Collection
<tb>90/10685HARMONY Collection
<tb>98/14719HARMONY Collection
<tb>97S99HARMONY Collection
<tb>97S100HARMONY Collection
<tb>825/96HARMONY Collection
<tb>842/96HARMONY Collection
<tb>N8-890/99HARMONY Collection
<tb>9805-01937HARMONY Collection
<tb>1Kreiswirth-1
<tb>29Kreiswirth-1
<tb>29060ATCC
<tb>35983ATCC
<tb>MRCNS (n = 4)35984ATCC
<tb>2514LSPQ
<tb>MA 52263LSPQ
<tb>6538ATCC
<tb>13301ATCC
<tb>25923ATCC
<tb>27660ATCC
<tb>29213ATCC
<tb>29247ATCC
<tb>29737ATCC
<tb>RN 11CDC
<tb>RN 3944CDC
<tb>RN 2442CDC
<tb>7605060113CDC
<tb>BM 4611Institut Pasteur
<tb>BM 3093Institut Pasteur
<tb>MSSA (n = 28)3511LSPQ
<tb>MA 5091LSPQ
<tb>MA 8849LSPQ
<tb>MA 8871LSPQ
<tb>MA 50607LSPQ
<tb>MA 50612LSPQ
<tb>MA 50848LSPQ
<tb>MA 51237LSPQ
<tb>MA 51351LSPQ
<tb>MA 52303LSPQ
<tb>MA 51828LSPQ
<tb>MA 51891LSPQ
<tb>MA 51504LSPQ
<tb>MA 52535LSPQ
<tb>MA 52783LSPQ
<tb>12228ATCC
<tb>14953ATCC
<tb>14990ATCC
<tb>15305ATCC
<tb>27836ATCC
<tb>27848ATCC
<tb>29070ATCC
<tb>29970ATCC
<tb>MSCNS (n = 17)29974ATCC
<tb>35539ATCC
<tb>35552ATCC
<tb>35844ATCC
<tb>35982ATCC
<tb>43809ATCC
<tb>43867ATCC
<tb>43958ATCC
<tb>49168ATCC
<tb><a>ATCC stands for "American Type Culture Collection".
LSPQ stands for "Laboratoire de Santé Publique du Québec".
CDC stands for "Center for Disease Control and Prevention".
Table 15. Clinical isolates used to test the sensitivity and/or specificity and/or ubiquity of the MRSA-specific PCR assays targeting MREJ sequences 
<tb>150Canada
<tb>10China
<tb>10Denmark
<tb>9Argentina
<tb>MRSA (n = 177)1Egypt
<tb>1Sweden
<tb>1Poland
<tb>3Japan
<tb>1France
<tb>208Canada
<tb>10China
<tb>MSSA (n = 224)4Japan
<tb>1USA
<tb>1Argentina
<tb>32Canada
<tb>3China
<tb>MRCNS (n = 38)1France
<tb>1Argentina
<tb>1USA
<tb>14UK
<tb>MSCNS (n = 17)3Canada
Table 16. Analytical sensitivity of tests performed on a standard thermocycler using the set of primers targeting MREP types i, ii, iii, iv and v (SEQ ID NOs.: 64, 66, 67, 79 and 80) developed in the present invention for the detection and identification of MRSA 
<tb>13370CCRI-8894 (i)10
<tb>ATCC 43300CCRI-175 (ii)5
<tb>9191CCRI-2086 (ii)10
<tb>35290CCRI-1262 (iii)5
<tb>352CCRI-1266 (iii)10
<tb>19121CCRI-8895 (iv)5
<tb>ATCC 33592CCRI-178 (iv)5
<tb>MA 50428CCRI-1311 (v)5
<tb>R991282CCRI-2025 (v)5
<tb><a>CCRI stands for "Collection of the Centre de Recherche en Infectiologie".
Table 17. Specificity and ubiquity tests performed on a standard thermocycler using the set of primers targeting MREP types i, ii, iii, iv and v (SEQ ID NO.: 64, 66, 67, 79 and 80) developed in the present invention for the detection and identification of MRSA 
<tb>MRSA - 35 strains<a>27 (77.1)8 (22.9)
<tb>MSSA - 44 strains13 (29.5)31 (70.5)
<tb>MRCNS - 9 strains*09 (100)
<tb>MSCNS - 10 strains*010 (100)
<tb><a>MRSA strains include the 20 strains listed in Table 3.
*Details regarding CNS strains: 
MRCNS :S. caprae(1)
S. cohni cohnii(1)
S. epidermidis(1)
S. haemolyticus(2)
S. hominis(1)
S. sciuri(1)
S. simulans(1)
S. warneri(1)
MSCNS :S. cohni(1)
S. epidermidis(1)
S. equorum (1)
S. haemolyticus(1)
S. lentus(1)
S. lugdunensis(1)
S. saccharolyticus(1)
S. saprophyticus(2)
S. xylosus(1)
Table 18. Analytical sensitivity of tests performed on the Smart Cycler® thermocycler using the set of primers targeting MREP types i, ii, iii, iv and v (SEQ ID NOs.: 64, 66, 67, 79 and 80) and molecular beacon probe (SEQ ID NO.: 84) developed in the present invention for the detection and identification of MRSA 
<tb>13370CCRI-8894 (i)2
<tb>ATCC 43300CCRI-175 (ii)2
<tb>9191CCRI-2086 (ii)10
<tb>35290CCRI-1262 (iii)2
<tb>352CCRI-1266 (iii)10
<tb>ATCC 33592CCRI-178 (iv)2
<tb>MA 51363CCRI-1331(iv)5
<tb>19121CCRI-8895 (iv)10
<tb>Z109CCRI-8903 (iv)5
<tb>45302CCRI-1263 (v)10
<tb>MA 50428CCRI-1311 (v)5
<tb>MA 50609CCRI-1312 (v)5
<tb>MA 51651CCRI-1325 (v)10
<tb>39795-2CCRI-1377 (v)10
<tb>R991282CCRI-2025 (v)2
<tb><a>CCRI stands for "Collection of the Centre de Recherche en Infectiologie".
Table 19. Specificity and ubiquity tests performed on the Smart Cycler® thermocycler using the set of primers targeting MREP types i, ii, iii, iv and v (SEQ ID NO. : 64, 66, 67, 79 and 80) and molecular beacon probe (SEQ ID NO. : 84) developed in the present invention for the detection of MRSA 
<tb>MRSA - 29 strains<a>21 (72.4)8 (27.6)
<tb>MSSA - 35 strains13 (37.1)22 (62.9)
<tb>MRCNS - 14 strains014 (100)
<tb>MSCNS - 10 strains010 (100)
<tb><a>MRSA strains include the 20 strains listed in Table 3.
Details regarding CNS strains:
MRCNS :S. epidermidis(1)
S. haemolyticus(5)
S. simulans(5)
S. warneri(3)
MSCNS :S. cohni cohnii(1)
S. epidermidis(1)
S. gallinarum(1)
S. haemolyticus(1)
S. lentus(1)
S. lugdunensis(1)
S. saccharolyticus(1)
S. saprophyticus(2)
S. xylosus (1)
Table 20. Analytical sensitivity of tests performed on the Smart Cycler® thermocycler using the set of primers targeting MREP types i, ii, iii, iv, v and vii (SEQ ID NOs.: 64, 66, 67, 79 and 80) and molecular beacon probe (SEQ ID NO.: 84) developed in the present invention for the detection and identification of MRSA 
<tb>13370CCRI-8894 (i)2
<tb>ATCC 43300CCRI-175 (ii)2
<tb>35290CCRI-1262 (iii)2
<tb>ATCC 33592CCRI-178 (iv)2
<tb>R991282CCRI-2025 (v)2
<tb>SE-41-1CCRI-9771 (vii)2
<tb><a>CCRI stands for "Collection of the Centre de Recherche en Infectiologie".
Table 21. Specificity and ubiquity tests performed on the Smart Cycler® thermocycler using the set of primers targeting MREP types i, ii, iii, iv, vi and vii (SEQ ID NOs.: 64, 66, 67, 79 and 80) and molecular beacon probe (SEQ ID NO.: 84) developed in the present invention for the detection and identification of MRSA 
<tb>MRSA - 23 strains<a>19 (82.6)4 (17.4)
<tb>MSSA - 25 strains13 (52)12 (48)
<tb>MRCNS - 26 strains026 (100)
<tb>MSCNS - 8 strains08 (100)
<tb><a>MRSA strains include the 20 strains listed in Table 3. Details regarding CNS strains:
MRCNS :S. capitis(2)
S. caprae(1)
S. cohnii(1)
S. epidermidis(9)
S. haemolyticus(5)
S. hominis(2)
S. saprophyticus(1)
S. sciuri(2)
S. simulans(1)
S. warneri(2)
MSCNS :S. cohni cohnii(1)
S. epidermidis(1)
S. haemolyticus(1)
S. lugdunensis(1)
S. saccharolyticus(1)
S.saprophyticus(2)
S. xylosus(1)
<img class="EMIRef" id="464672256-ib0001" />
<img class="EMIRef" id="464672256-ib0002" />


[0113] In view of the foregoing, it will be appreciated that the invention described hereininter aliarelates to the following items:
1. A method to detect the presence of a methicillin-resistantStaphylococcus aureus(MRSA) strain in a sample, said MRSA strain being resistant because of the presence of an SCCmecinsert containing amecAgene, said SCCmecbeing inserted in bacterial nucleic acids thereby generating a polymorphic right extremity junction (MREJ), said method comprising the step of annealing the nucleic acids of the sample with a plurality of probes and/or primers, characterized by:
(i) said primers and/or probes are specific for MRSA strains and capable of annealing with polymorphic MREJ nucleic acids, said polymorphic MREJ comprising MREJ types i to x; and
(ii) said primers and/or probes altogether can anneal with at least four MREJ types selected from MREJ types i to x.2. The method of item 1, wherein the primers and/or probes are all chosen to anneal under common annealing conditions.
3. The method of item 2, wherein the primer and/or probes are placed altogether in the same physical enclosure.
4. The method of any one of items 1 to 3, wherein the primers and/or probes have at least 10 nucleotides in length and are capable of annealing with MREJ types i to iii, defined in any one of SEQ ID NOs: 1, 20, 21, 22, 23, 24, 25, 41; 199 ; 2, 17, 18, 19, 26, 40, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 197 ; 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 104, 184, 198 ;
and with one or more of MREJ types iv to ix, having SEQ ID NOs: 42, 43, 44, 45, 46, 51 ; 47, 48, 49, 50 ; 171 ; 165, 166 ; 167 ; 168.
5. The method of any one of items 1 to 4, wherein the primers and/or probes altogether can anneal with said SEQ ID NOs of MREJ types i to ix.
6. The method of any one of items 1 to 5, wherein said primers and/or probes have the following sequences SEQ ID NOs:
<tb>66, 100, 101, 105, 52, 53, 54, 55,for the detection of MREJ type i
<tb>56, 57, 64, 71, 72, 73, 74, 75, 76,
<tb>70, 103, 130; 132, 158, 159, 59,
<tb>62, 126, 127, 128, 129, 131, 200,
<tb>201,60,61,63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>66, 97, 99, 100, 101, 106, 117,for the detection of MREJ type ii
<tb>118, 124, 125, 52, 53, 54, 55, 56, 57
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60,61,63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>67, 98, 102, 107, 108for the detection of MREJ type iii
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 132, 158, 159
<tb>58,
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60,61,63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>79, 77, 145, 147for the detection of MREJ type iv
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60, 61, 63
<tb>68
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85,86,87,88,89
<tb>65, 80, 146, 154, 155for the detection of MREJ type v
<tb>64, 71, 72, 73, 74, 75, 76,
<tb>70, 103, 130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60,61,63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>202, 203, 204for the detection of MREJ-type vi
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60,61,63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>112, 113, 114, 119, 120, 121, 122for the detection of MREJ type vii
<tb>, 123, 150, 151, 153
<tb>64, 71, 72, 73, 74, 75, 76, 70, 103,
<tb>130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60,61,63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>115, 116, 187, 188, 207, 208for the detection of MREJ type viii
<tb>64, 71, 72, 73, 74, 75, 76, 70,
<tb>103, 130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60,61,63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 89
<tb>109, 148, 149, 205, 206for the detection of MREJ type ix.
<tb>64, 71, 72, 73, 74, 75, 76
<tb>70, 103, 130, 132, 158, 159
<tb>59,62
<tb>126, 127
<tb>128, 129, 131, 200, 201
<tb>60,61,63
<tb>32, 83, 84, 160, 161, 162, 163, 164
<tb>85, 86, 87, 88, 897. The method of item 6, wherein primer pairs have the nucleotide sequence which are defined in SEQ ID NOs :
<tb>64/66, 64/100, 64/101; 59/52,for the detection of type i MREJ
<tb>59/53, 59/54, 59/55, 59/56, 59/57,
<tb>60/52, 60/53, 60/54, 60/55, 60/56
<tb>60/57, 61/52, 61/53, 61/54, 61/55
<tb>61/56, 61/57, 62/52, 62/53, 62/54
<tb>62/55, 62/56, 62/57, 63/52, 63/53
<tb>63/54, 63/55, 63/56, 63/57
<tb>64/66, 64/97, 64/99, 64/100, 64/101for the detection of type ii MREJ
<tb>59/52, 59/53, 59/54, 59/55, 59/56,
<tb>59/57, 60/52, 60/53, 60/54, 60/55,
<tb>60/56, 60/57, 61/52, 61/53, 61/54,
<tb>61/55, 61/56, 61/57, 62/52, 62/53,
<tb>62/54, 62/55, 62/56, 62/57, 63/52
<tb>63/53, 63/54, 63/55, 63/56, 63/57
<tb>64/67, 64/98, 64/102 ; 59/58,for the detection of type iii MREJ
<tb>60/58, 61/58, 62/58, 63/58
<tb>64/79for the detection of type iv MREJ
<tb>64/80for the detection of type v MREJ
<tb>64/204for the detection of type vi MREJ
<tb>64/112, 64/113for the detection of type vii MREJ
<tb>64/115, 64/116for the detection of type viii MREJ
<tb>64/109for the detection of type ix MREJ8. The method of item 7, further comprising probes having the following sequences: SEQ ID NOs: 32, 83, 84, 160, 161, 162, 163,164 for the detection of MREJ types i to ix.
9. The method of any one of items 6 to 8, wherein said primers and probes have the following nucleotide sequences:
vii) SEQ ID NOs: 64, 66, 84, 163, 164 for the detection of MREJ type i
viii) SEQ ID NOs: 64, 66, 84, 163, 164 for the detection of MREJ type ii
ix) SEQ ID NOs: 64, 67, 84, 163, 164 for the detection of MREJ type iii
x) SEQ ID NOs: 64, 79, 84, 163, 164 for the detection of MREJ type iv
xi) SEQ ID NOs: 64, 80, 84, 163, 164 for the detection of MREJ type v
xii) SEQ ID NOs: 64, 112, 84, 163, 164 for the detection of MREJ type vii.10. The method of any one of items 1 to 8, wherein said probes and primers are used together.
11. The method of item 9 or 10, wherein said probes and/or primers are used together in the same physical enclosure.
12. A method for typing a MREJ of a MRSA strain, which comprises the steps of:
reproducing the method of any one of items 1 to 11 with primers and/or probes specific for a determined MREJ type, and detecting an annealed probe and/or primer as an indication of the presence of a determined MREJ type.13. A nucleic acid selected from:
vii)SEQ ID NOs: 42, 43, 44, 45, 46, 51 for sequence of MREJ type iv ;
viii) SEQ ID NOs: 47, 48, 49, 50 for sequence of MREJ type v ;
ix) SEQ ID NOs: 171 for sequence of MREJ type vi ;
x) SEQ ID NOs: 165, 166 for sequence of MREJ type vii ;
xi) SEQ ID NOs: 167 for sequence of MREJ type viii ;
xii)SEQ ID NOs: 168 for sequence of MREJ type ix.14. An oligonucleotide of at least 10 nucleotides in length which hybridizes with the nucleic acid of item 13 and which hybridizes with one or more MREJ of types selected from iv to ix.
15. An oligonucleotide pair which has the nucleotide sequences defined in any one of SEQ ID NOs:
<tb>64/66, 64/100, 64/101; 59/52,for the detection of type i MREJ
<tb>59/53, 59/54, 59/55, 59/56, 59/57,
<tb>60/52, 60/53, 60/54, 60/55, 60/56
<tb>60/57, 61/52, 61/53, 61/54, 61/55
<tb>61/56, 61/57, 62/52, 62/53, 62/54
<tb>62/55, 62/56, 62/57, 63/52, 63/53
<tb>63/54, 63/55, 63/56, 63/57
<tb>64/66, 64/97, 64/99, 64/100, 64/101for the detection of type ii MREJ
<tb>59/52, 59/53, 59/54, 59/55, 59/56,
<tb>59/57, 60/52, 60/53, 60/54, 60/55,
<tb>60/56, 60/57, 61/52, 61/53, 61/54,
<tb>61/55, 61/56, 61/57, 62/52, 62/53,
<tb>62/54, 62/55, 62/56, 62/57, 63/52
<tb>63/53, 63/54, 63/55, 63/56, 63/57
<tb>64/67, 64/98, 64/102 ; 59/58,for the detection of type iii MREJ
<tb>60/58, 61/58, 62/58, 63/58
<tb>64/79for the detection of type iv MREJ
<tb>64/80for the detection of type v MREJ
<tb>64/204for the detection of type vi MREJ
<tb>64/112, 64/113for the detection of type vii MREJ
<tb>64/115, 64/116for the detection of type viii MREJ
<tb>64/109for the detection of type ix MREJ16. An oligonucleotide which has the nucleotide sequence defined in any one of SEQ ID NOs: 32, 83, 84, 160, 161, 162, 163, 164.
17. A composition of matter comprising primers and/or probes, the nucleotide sequences of which have at least 10 nucleotides in length which hybridize with any nucleic acid defined in item 13, and which hybridize with one or more MREJ of types selected from iv to ix.
18. The composition of item 17, which further comprises primers and/or probes, which hybridize with one or more MREJ of types selected from i to iii.
19. The composition of item 18 or 19, wherein the primers pairs have the nucleotide sequences defined in SEQ ID NOs:
<tb>64/66, 64/100, 64/101; 59/52,for the detection of type i MREJ
<tb>59/53, 59/54, 59/55, 59/56, 59/57,
<tb>60/52, 60/53, 60/54, 60/55, 60/56
<tb>60/57, 61/52, 61/53, 61/54, 61/55
<tb>61/56, 61/57, 62/52, 62/53, 62/54
<tb>62/55, 62/56, 62/57, 63/52, 63/53
<tb>63/54, 63/55, 63/56, 63/57
<tb>64/66, 64/97, 64/99, 64/100, 64/101for the detection of type ii MREJ
<tb>59/52, 59/53, 59/54, 59/55, 59/56,
<tb>59/57, 60/52, 60/53, 60/54, 60/55,
<tb>60/56, 60/57, 61/52, 61/53, 61/54,
<tb>61/55, 61/56, 61/57, 62/52, 62/53,
<tb>62/54, 62/55, 62/56, 62/57, 63/52
<tb>63/53, 63/54, 63/55, 63/56, 63/57
<tb>64/67, 64/98, 64/102 ; 59/58,for the detection of type iii MREJ
<tb>60/58, 61/58, 62/58, 63/58
<tb>64/79for the detection of type iv MREJ
<tb>64/80for the detection of type v MREJ
<tb>64/204for the detection of type vi MREJ
<tb>64/112, 64/113for the detection of type vii MREJ
<tb>64/115, 64/116for the detection of type viii MREJ
<tb>64/109for the detection of type ix MREJ20. The composition of item 18, which further comprises probes, which SEQ ID NOs are: 32, 83, 84, 160, 161, 162, 163, 164.
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