WO2020142852A1 - Novel assay for mrsa detection directly from clinical samples - Google Patents

Abstract

Disclosed are primers for detecting a methicillin-resistant Staphylococcus aureus (MRSA) DNA in a sample from a subject, wherein a signal indicative of or∫X and a signal indicative of mecA are generated if MRSA DNA is present. A method of detecting MRSA using the primers and a kit comprising the primers are also disclosed.

Description

NOVEL ASSAY FOR MRSA DETECTION DIRECTLY FROM CLINICAL SAMPLES

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to United States provisional patent application number US 62/791 ,245, filed January 1 1 , 2019, the entire contents of which is hereby incorporated by reference.

FIELD

[0002] The present disclosure relates generally to a novel assay for direct MRSA detection designed to circumvent the deficits of commercially available assays

BACKGROUND

[0003] Methicillin-resistant Staphylococcus aureus (MRSA) strains emerged shortly after clinical methicillin use (1 , 2) through acquisition of the methicillin resistance gene mecA, which is carried on a mobile genetic element, staphylococcal cassette chromosome mec ( SCCmec ) (3, 4). Strains of MRSA have spread and become established as major nosocomial pathogens worldwide (5-9), and more recently have emerged as a major cause of community-acquired infections (10, 1 1). While many SA strains remain susceptible to penicillinase-stable penicillins, such as oxacillin and methicillin and other antimicrobial agents, MRSA is more drug-resistant than their methicillin-susceptible SA (MSSA) counterparts (12). Although 1 st generation cephalosporins (eg. cefazolin and cephalexin) and isoxazolyl penicillins (oxacillin, cloxacillin) are still the agents of choice for treatment of MSSA infection, MRSA is resistant to most b-lactam agents, including cephalosporins (with the exception of the new advanced 5th generation agents, ceftibiprole and ceftaroline) and carbapenems.

With limited treatment options, infection control practices such as isolation are necessary to help control the spread of MRSA. The timely detection of MRSA is, consequently, crucial for optimal management of this pathogen, not only for optimal patient outcomes but also for limiting the nosocomial spread of this organism.

[0004] Early detection is, however, complicated by the fact that in clinical samples pathogenic SA is often mixed with putatively less pathogenic coagulase negative staphylococci (CoNS), both of which can harbour mecA. Conventional methods for the detection of MRSA in clinical microbiology laboratories depends on growth of the organism with selective media, which is time consuming and generally requires 2-3 days. Nucleic acid amplification methods are also used to discriminate MRSA from MSSA and CoNS, however, traditional genetic identification methods require PCR amplification of a pure bacterial culture This is because both SA and CoNS can carry mecA, and it is not possible to determine if the mecA positive originates from MRSA, or from a methicillin- resistant CoNS (M, R-CoNS) that is also present in the clinical sample.

[0005] Currently, two FDA-approved commercially available PCR based assays are widely used to detect MRSA directly from clinical samples, including IDI- MRSA/GeneOhm MRSA (BD Diagnostics, Franklin Lakes, NJ, USA) and GeneXpert MRSA (Cepheid, Sunnyvale, CA, USA). Both assays are similar, using quantitative realtime PCR-based methods targeting the 3’ end of the orfX gene in SA, in conjunction with the right extremity of SCCmec, but not targeting mecA directly. However, both assays are problematic in that they produce false negative and false positive results. False negatives arise from the inability to detect new, variant and nontypeable SCCmec cassettes with the existing primers, and false positives from the presence of SCC-like elements that do not contain mecA, which are incorrectly amplified (13-17). Moreover, any emerging variant SCCmec new types will not be detected either.

SUMMARY

[0006] In one aspect there is provided a first long range primer comprising a polynucleotide molecule of Sequence (I),

[capture moiety]-[first tail]-[orfX] (I).

[0007] In one example, said [capture moiety] comprises a polynucleotide that is labelled with biotin, beads coated/labelled with biotin, other fluorescent labels, or tags.

[0008] In one example, said [first tail] comprises a polynucleotide sequence that does not match a known bacterial genome.

[0009] In one example, said [first tail] is 20 - 35 base pairs long, or 20 base pairs long or 21 base pairs long.

[0010] In one example, said [orfX] comprises a polynucleotide that anneals to at least of a portion of the orfX gene.

[0011] In one example, wherein [capture moiety]-[first tail]-[orfX] is SA-3e (SEQ ID

NO: 1).

[0012] In one aspect there is provided a first long range primer comprising a polynucleotide molecule of Sequence (la),

[orfX] (la). [0013] In one example, wherein said [first tail] comprises a polynucleotide sequence that does not match a known bacterial genome.

[0014] In one example, said [first tail] is 20 - 35 base pairs long, or 20 base pairs long or 21 base pairs long.

[0015] In one example, said [orfX] comprises a polynucleotide that anneals to at least of a portion of the orfX gene.

[0016] In one example, wherein [first tail]-[orfX] is SA-3e MOD (SEQ ID NO: 13).

[0017] In one aspect there is provided a second long range primer comprising a polynucleotide molecule of Sequence (II),

[second tail]-[mecA] (II).

[0018] In one example, said [second tail] comprises a polynucleotide sequence that does not match a known bacterial genome.

[0019] In one example, wherein said [second tail] is 20 - 35 base pairs long, or 20 base pairs long or 21 base pairs long.

[0020] In one example, said [mecA] comprises a polynucleotide that anneals to at least portion of the meAc gene.

[0021] In one example, wherein [second tail]-[mecA] is Umec-F (SEQ ID NO: 2).

[0022] In one aspect there is provided a third long range primer comprising a polynucleotide molecule of Sequence (III),

[second tail]-[reversed Umec] (III).

[0023] In one example, said [second tail] comprises a polynucleotide sequence that does not match a known bacterial genome.

[0024] In one example, said [second tail] is 20 - 35 base pairs long, or 20 base pairs long or 21 base pairs long.

[0025] In one example, wherein said [reversed Umec] comprises a polynucleotide primer that anneals to at least a portion of reversed Umec of SCCmecX.

[0026] In one example, wherein the [second tail]-[reversed Umec] is Umec-FX is

(SEQ ID NO: 3).

[0027] In one aspect there is provided a fourth primer comprising a polynucleotide molecule of Sequence (IV),

[second orfX] (IV).

[0028] In one example, wherein [second orfX] comprises a polynucleotide primer that anneals to at least a portion of the orfX gene.

[0029] In one example, wherein [second orfX] is Arb3 (SEQ ID NO: 4). [0030] In one aspect there is provided a fifth primer comprising a polynucleotide molecule of Sequence (V),

[right side first tail] (V).

[0031] In one example, [right side first tail] comprises a polynucleotide primer that anneals to a right side of the first tail of the first long range primer.

[0032] In one example, wherein [right side first tail] is SA-4 (SEQ ID NO: 5).

[0033] In one aspect there is provided a sixth primer comprising a polynucleotide molecule of Sequence (VI),

[first probe] (VI).

[0034] In one example, wherein [first probe] comprises a polynucleotide primer that anneals to In one example, wherein [first probe] comprises at least one quencher and/or at least on fluorescent molecule.

[0035] In one example, wherein the at least one quencher is ZEN, ABkFQ, and/or

TAMRA.

[0036] In one example, wherein the at least fluorescent moiety is Fam, Tet, and/or

ROX

[0037] In one example, wherein [first probe] is SA-HEX-1 (SEQ ID NO: 6) or SA-

HEX-1 MOD (SEQ ID NO: 14).

[0038] In one aspect there is provided a seventh primer comprising a

polynucleotide molecule of Sequence (VII),

[UmecF/UmecFX] (VII).

[0039] In one example, wherein [UmecF/UmecFX] comprises a polynucleotide primer that anneals to at least a portion of [second tail] of the second long range primer and the third long range primer.

[0040] In one example, wherein [UmecF/UmecFX] is Arb2 (SEQ ID NO: 7).

[0041] In one aspect there is provided an eighth primer comprising a

polynucleotide molecule of Sequence (VIII),

[UmecR primer] (VIII).

[0042] In one example, wherein [UmecR primer] comprises a polynucleotide primer that anneals to at least a portion of the mecA gene in the typical orientation.

[0043] In one example, wherein [UmexR primer] is Umec-R (SEQ ID NO: 8).

[0044] In one aspect there is provided a ninth primer comprising a polynucleotide molecule of Sequence (IX),

[Umec-RX primer] (IX). [0045] In one example, wherein [Umec-RX primer] comprises a polynucleotide primer that anneals to at least a portion of the mecA gene in the reversed orientation.

[0046] In one example, wherein [Umec-RX primer] is Umec-RX (SEQ ID NO: 9).

[0047] In one aspect there is provided a tenth primer comprising a polynucleotide molecule of Sequence (X),

[mec-FAM-2 detection probe] (X).

[0048] In one example, wherein [mec-FAM-2 detection probe] comprises a polynucleotide primer that anneals to at least a portion of the meAc gene.

[0049] In one example, wherein [mec-FMA-2 detection probe] comprises at least one quencher and/or at least on fluorescent molecule.

[0050] In one example, wherein the at least one quencher is ZEN, ABkFQ, and/or

TAMRA.

[0051] In one example, wherein the least one fluorescent moiety is FAM, Tet, and/or ROX.

[0052] In one example, wherein [mec-FAM-2 detection probe] is m-eFcAM-2

(SEQ ID NO: 10) or [mec-MOD-2] (SEQ ID NO: 15).

[0053] In one aspect there is provided an eleventh primer comprising a polynucleotide molecule of Sequence (XI),

[mec-FAM-3 detection probe] (XI).

[0054] In one example, wherein [mec-FAM-3 detection primer] comprises a polynucleotide primer that anneals to at least a portion of the meAc gene.

[0055] In one example, wherein [mec-FAM-3 detection primer] comprises at least one quencher and/or at least one fluorescent molecule.

[0056] In one example, wherein the at least one quencher is ZEN, ABkFQ, and/or

TAMRA.

[0057] In one example, wherein the at least one fluorescent moiety is FAM, Tet, and/or ROX.

[0058] In one example, wherein [mec-FAM-3 detection probe] is m-eFcAM-3

(SEQ ID NO: 1 1) or [mec-MOD-3] (SEQ ID NO: 16)..

[0059] In one aspect there is provided a twelfth primer comprising a

polynucleotide molecule of Sequence (XII),

[mec-FAM-4 detection probe] (XII).

[0060] In one example, wherein [mec-FAM-3 detection primer] comprises a polynucleotide primer that anneals to at least a portion of the meAc gene. [0061] In one example, wherein [mec-FAM-3 detection primer] comprises at least one quencher and/or at least on fluorescent molecule.

[0062] In one example, wherein the at least one quencher is ZEN, ABkFQ, and/or

TAMRA.

[0063] In one example, wherein wherein the least one fluorescent moiety is FAM,

Tet, and/or ROX.

[0064] In one example, wherein [mec-FAM-4 detection probe] is m-eFcAM-4

(SEQ ID NO: 12) or [mec-MOD-4] (SEQ ID NO: 17)..

[0065] In one aspect there is provided a method of detecting in a sample from a subject a methicillin-resistant Staphylococcus aureus (MRSA), DNA, comprising:

[0066] a) performing a first amplification reaction, comprising, combining said sample with a first long range primer of any one of claims 1 to 11 , a second long range primer of any one of claims 12 to 16, and a third long range primer of any one of claims 17 to 21 , under amplification conditions to obtain a first amplification product,

[0067] b) performing a second amplification reaction, comprising, combining a portion of said first amplification product with a fourth primer of any one of claims 22 to 24, a fifth primer of any one of claims 25 to 27, and a sixth primer of any one of claims 28 to 33, under amplification conditions to detect a signal indicative of orfX, if MRSA DNA is present in said sample, and

[0068] c) performing a third amplification reaction, comprising combining a portion of said first amplification product with a seventh primer of any one of claims 34 to 36, an eighth primer of any one of claims 37 to 39, a ninth primer of any one of claims 40 to 42, a tenth primer of any one of claims 43 to 48, an eleventh primer of any one of claims 49 to 54, and a twelfth primer of any one of claims 55 to 60, under amplification conditions to detect a signal indicative of mecA, if MRSA DNA is present in said sample.

[0069] In one example, further comprising a step of enriching said amplification product of step a) prior to step b) and step c).

[0070] In one example, wherein said subject is a human.

[0071] In one aspect there is provided a kit comprising: a containing, and/or a first long range primer of any one of claims 1 to 1 1 , and/or a second long range primer of any one of claims 12 to 16, and/or a third long range primer of any one of claims 17 to 21 , a fourth primer of any one of claims 22 to 24, and/or a fifth primer of any one of claims 25 to 27, and/or a sixth primer of any one of claims 28 to 33, a seventh primer of any one of claims 34 to 36, and/or an eighth primer of any one of claims 37 to 39, and/or a ninth primer of any one of claims 40 to 42, and/or a tenth primer of any one of claims 43 to 48, and/or an eleventh primer of any one of claims 49to 54, and/or a twelfth primer of any one of claims 55 to 60.

BRIEF DESCRIPTION OF THE FIGURES

[0072] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0073] Figure 1. A new scheme for the MRSA directly clinical sample detection, designed to circumvent deficits in commercially available assays. Primers for the initial long range (LR) PCR are located in the orfX and mecA genes (light grey and thick grew arrows respectively), and contain tail sequences (indicated by double and dashed lines) that are unique, and function as templates for Round 2 primers (indicated by double line and dashed arrows). Primer SA-3e is biotin labeled (grey crescent), allowing the LR-PCR product to be captured by streptavidin coated magnetic beads (black orb), and concentrated/purified. The Round 2 real-time PCR reactions are detected with probes specific to SA’s orfX (vertical stripe) and mecA (horizontal stripe).

[0074] Figure 2. orfX and mecA reaction amplification curves show that all

SCCmec types are detected. A) Detection of SCCmec types with the orfX reaction, using fluorophore HEX, shows that all types pass the threshold and have C(t) values between cycles 1 1 and 19, while the no template control (NTC) remains negative below the threshold. B) Detection of SCCmec types with the mecA reaction, using fluorophore FAM, shows that all types pass the threshold and have C(t) values between cycles 13 and 27, while the no template control (NTC) remains negative below the threshold.

DETAILED DESCRIPTION

[0075] In some aspects there is described primers and/or methods for amplifying and/or detecting MRSA polynucleotides in a sample from a subject.

[0076] In one example there is described primers and/or methods for the amplifying and/or detection of mecA polynucleotides from MRSA in a sample from a subject.

[0077] In another example there is described primers and/or methods for amplifying and/or detecting mecA polynucleotides and orfX polynucleotides from MRSA in a sample from a subject.

[0078] In some examples, amplification and/or detection methods may include the polymerase chain reaction (“PCR”), including but not limited to, Long Range polymerase chain reaction (“LR-PCR”) and real-time PCR (“RT-PCR”). [0079] Staphylococcus aureus is a gram-positive bacterium which can cause diseases including skin infection, respiratory infection, meningitis, endocarditis, toxic shock syndrome, and sepsis. It is one of the most common nosocomial infections and is increasing in frequency in neonatal intensive care units. Multi-drug resistant strains (including methicillin-resistant S. aureus; MRSA) are increasingly common.

[0080] MRSA includes, but is not limited to, hospital-acquired MRSA (HA-MRSA) or community-acquired MRSA (CA-MRSA).

[0081] The term“subject”, as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject may be an infant, a child, an adult, or elderly. In a specific example, the subject is a human.

[0082] In some example, a subject is suspected of having MRSA or at risk of having or developing MRSA.

[0083] A subject at risk of having MRSA infection is a subject that is predisposed to develop an infection. Such a subject can include, for example, a subject with a known or suspected exposure to an infectious organism or agent, such as MRSA. A subject at risk of having an infection also can include a subject with a condition associated with impaired ability to mount an immune response to an infectious organism or agent.

[0084] The term“infection” as used herein, refers to a disease or condition attributable to the presence in a host of a foreign organism or agent that reproduces within the host. Infections typically involve breach of a normal mucosal or other tissue barrier by an infectious organism or agent. In a specific example, an infection is an MRSA infection.

[0085] The term“sample” or“biological sample”, as used herein, refers to animal or human samples including, without limitation, any biological fluid (blood, bone marrow, plasma, serum, bronchoalveolar washing fluid, urine, nasal secretion, ear secretion, urethral secretion, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, sputum, lymph, plasma, ejaculate, lung aspirate, etc.), cell, tissue, organ or portion thereof that contains DNA. A sample can be obtained by conventional methods, using processes known in the state of the art by the person skilled in the art. In some examples, a sample is used directly (e.g., fresh or frozen), or can be manipulated prior to use, for example, by extraction (for example of nucleic acids), fixation (e.g., using formalin) and/or embedding in wax (such as FFPE tissue samples). Samples also include all samples useful for detection of a pathogen in an environment (such as a clinic or hospital), including but not limited to a water or fluid sample, a food sample, or a surface swab.

[0086] The terms“polynucleotides”, "nucleic acid" and“oligonucleotides” refers to biopolymers of nucleotides and, unless the context indicates otherwise, includes modified and unmodified nucleotides, and both DNA and RNA. Typically, the methods as described herein are performed using DNA as the nucleic acid template for amplification.

It will be appreciated that nucleic acid whose nucleotide is replaced by an artificial derivative or modified nucleic acid from natural DNA or RNA is also included in the nucleic acid of the present invention insofar as it functions as a template for synthesis of complementary chain.

[0087] The nucleic acid or“target nucleic acid” is generally contained in a biological sample. In some examples, MRSA DNA may be the target nucleic acid.

[0088] The term“primer”,“polynucleotide primer” refers to a short polynucleotide that satisfies the requirements that it is able to form complementary base pairing sufficient to anneal to a desired nucleic acid template for use in the methods herein, including amplification reactions such as PCR, LR-PCR, and RT-PCR. Typically, at least two primers are utilized in an amplification reaction. In some examples, more than two primers are utilized in an amplification reaction. Thus, the term“primer” and its variants can include any single stranded nucleic acid molecule (regardless of length) that, once hybridized to a complementary nucleic acid sequence, can prime nucleic acid synthesis. Typically, such nucleic acid synthesis occurs in a template-dependent fashion, and nucleotides are polymerized onto at least one end of the primer during such nucleic acid synthesis. The term“primer extension” and its variants, as used herein, when used in reference to a given method, relates to any method for catalyzing nucleotide incorporation onto a terminal end of a nucleic acid molecule. Typically but not necessarily such nucleotide incorporation occurs in a template-dependent fashion.

[0089] A primer may also be modified by attachment of one or more chemical moieties including but not limited to, biotin, a fluorescent tag, a phosphate, or a chemically reactive group.

[0090] As used herein the term“amplification” and its variants includes any process for producing multiple copies or complements of at least some portion of a polynucleotide, said polynucleotide typically being referred to as a“template” or, in some cases, as a“target.” The template (or target) polynucleotide can be single stranded or double stranded. Amplification of a given template can result in the generation of a population of polynucleotide amplification products. [0091] It will be appreciated that the primer backbone is not necessarily limited to the one via phosphodiester linkages. For example, it may be composed of a

phosphothioate derivative having S in place of O as a backbone or a peptide nucleic acid based on peptide linkages. The bases may be those capable of complementary base pairing.

[0092] The oligonucleotides as used herein may function not only as the origin of synthesis but also as a template for synthesis of complementary chain. The term polynucleotide includes oligonucleotides, which have a relatively short chain length. A primer need not be fully complementary in order to anneal to a binding site on a polynucleotide.

[0093] The primers are generally isolated.

[0094] The terms isolated as used herein generally refers to a biological component (such as a nucleic acid) that has been substantially separated or purified away from biological or other components. Nucleic acids that have been“isolated” include nucleic acids purified by standard purification methods. The term also embraces nucleic acids prepared by recombinant expression in a host cell and subsequently purified, as well as chemically synthesized nucleic acid molecules. Isolated does not require absolute purity, and can include nucleic acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated. An isolated nucleic acid may be in solution (e.g., water or an aqueous solution) or dried.

[0095] Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle, as opposed to endpoint detection. The real-time progress of the reaction can be viewed in some systems.

Typically, real-time PCR uses the detection of a fluorescent reporter. Typically, the fluorescent reporter's signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.

[0096] In some examples, the fluorescently-labeled probes rely upon fluorescence resonance energy transfer (FRET), or in a change in the fluorescence emission wavelength of a sample, as a method to detect hybridization of a DNA probe to the amplified target nucleic acid in real-time. [0097] In some examples, the fluorescently-labeled DNA probes used to identify amplification products have spectrally distinct emission wavelengths, thus allowing them to be distinguished within the same reaction tube, for example in multiplex PCR, such as a multiplex real-time PCR. In some embodiments, the probes and primers disclosed herein are used in multiplex real-time PCR.

[0098] In some examples, there is described an assay comprising two PCR steps

(Figurel), including an initial long range (LR-) PCR reaction (Round 1), the product of which acts as the template for the second round real-time PCR reactions (Round 2). Forward and reverse primers for the initial LR- PCR reaction are located in the orfX and mecA genes, creating a PCR product that ranges in size from 2.3 kb to 42.3 kb. By simultaneously targeting both of these regions, the assay was designed to eliminate templates that do not contain the mecA gene, as well as anything that is not

Staphylococcus aureus. The second round real-time PCR was designed as 2 separate reactions; one to detect the orfX gene, and the other to detect the mecA gene.

[0099] Round one long range PCR reaction and primers

[00100] In one example there is described a first long range primer comprising a polynucleotide molecule of Sequence (I),

[00101] [capture moiety]-[first tail]-[orfX (I).

[00102] The reaction product nucleic acid molecules of the round one long range PCR reaction may be enriched of isolated using a capture moiety.

[00103] In some examples, a capture moiety selectively binds to a binding agent. For example, the capture moiety can include a first member of a binding pair, and the binding agent can include a second member of the same binding pair. In some embodiments, the capture oligonucleotide includes a biotin capture moiety, and the binding agent is a streptavidin-containing support.

[00104] As used herein, the term“binding partners” includes two molecules, or portions thereof, which have a specific binding affinity for one another and typically will bind to each other in preference to binding to other molecules. Typically but not necessarily some or all of the structure of one member of a specific binding pair is complementary to some or all of the structure possessed by the other member, with the two members being able to bind together specifically by way of a bond between the complementary structures, optionally by virtue of multiple noncovalent attractions.

[00105] In some examples, molecules that function as binding partners may include, but are not limited to, biotin (and its derivatives) and their binding partner avidin moieties, streptavidin moieties (and their derivatives); His-tags which bind with nickel, cobalt or copper; cysteine, histidine, or histidine patch which bind Ni-NTA; maltose which binds with maltose binding protein (MBP); lectin-carbohydrate binding partners; calcium- calcium binding protein (CBP); acetylcholine and receptor-acetylcholine; protein A and binding partner anti-FLAG antibody; GST and binding partner glutathione; uracil DNA glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor) protein; antigen or epitope tags which bind to antibody or antibody fragments, particularly antigens such as digoxigenin, fluorescein, dinitrophenol or bromodeoxyuridine and their respective antibodies; mouse immunoglobulin and goat anti-mouse immunoglobulin; IgG bound and protein A; receptor-receptor agonist or receptor antagonist; enzyme-enzyme cofactors; enzyme-enzyme inhibitors; and thyroxine-cortisol.

[00106] An avidin moiety may include an avidin protein, as well as any derivatives, analogs and other non-native forms of avidin that can bind to biotin moieties. Other forms of avidin moieties include native and recombinant avidin and streptavidin as well as derivatized molecules, e.g. nonglycosylated avidins, N-acyl avidins and truncated streptavidins. For example, avidin moiety includes deglycosylated forms of avidin, bacterial streptavidins produced by Streptomyces (e.g., Streptomyces avidinii), truncated streptavidins, recombinant avidin and streptavidin as well as to derivatives of native, deglycosylated and recombinant avidin and of native, recombinant and truncated streptavidin, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyl and N-succinyl avidin, and the commercial products ExtrAvidin™, Captavidin™, Neutravidin™ and Neutralite Avidin™.

[00107] In a specific example [capture moiety] is a polynucleotide encoding biotin.

[00108] The [first tail] comprises a polynucleotide sequence that does not match a known bacterial genome. In a specific example, the [first tail] is 20 base pairs or 21 base pairs.

[00109] The [orfX\ is a polynucleotide that anneals to at least a portion of the orfX gene.

[00110] In a specific example, a first long range primer is SA-3e.

[00111] In other example, the first long range primer is least 80% identical, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SA-3e.

[00112] In one example there is described a second long range primer comprising a polynucleotide molecule of Sequence (II),

[00113] [second tail]-[mecA] (II). [00114] The [second tail] comprises a polynucleotide sequence that does not match a known bacterial genome. In a specific example, the [first tail] is 20 base pairs or 21 base pairs.

[00115] The [mecA] is a polynucleotide that anneals to at least portion of the mecA gene.

[00116] In a specific example, a second long range primer is Umec-F.

[00117] In other example, the second long range primer is 80% identical, such as

80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Umec-F.

[00118] In one example there is described a third long range primer comprising a polynucleotide molecule of Sequence (III),

[00119] [second tail]-[reversed Umec] (II)

[00120] The [second tail] comprises a polynucleotide sequence that does not match a known bacterial genome.

[00121] In a specific example, the [first tail] is 20 base pairs or 21 base pairs.

[00122] The [reversed Umec] is a polynucleotide primer that anneals to at least a portion of reversed Umec of SCCmecX.

[00123] In a specific example, a third long range primer is Umec-FX.

[00124] In other example, the third long range primer is least 80% identical, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Umec-FX.

[00125] Thus, in a specific example, forward and reverse primers for the initial LR- PCR reaction are located in the orfX and mecA genes, creating a PCR product that ranges in size from 2.3 kb to 42.3 kb. By simultaneously targeting both of these regions, the assay was designed to eliminate templates that do not contain the mecA gene, as well as anything that is not Staphylococcus aureus. Primer Umec-F targets a conserved sequence in the mecA/mecC gene, present in all SCCmec types except type X. Since the orientation of mecA in SCCmec X is reversed compared to the other types, primer Umec- FX was included to effectively detect that type, as well as any future SCCmec types with the reversed mecA orientation. Primer SA-3e, on the other hand, targets a region that is specific to the S. aureus orfX gene and is universally found in all S. aureus with available sequences. The target sequence differs significantly enough from that found in CoNS, thereby preventing amplification from CoNS orfX genes. Primer SA-3e also carries a 5’ biotin label, allowing the LR-PCR products to be captured with streptavidin coated magnetic beads, thereby concentrating them and purifying them of residual LR-PCR .eaction components. Magnetic beads containing captured LR-PCR product from Round 1 are added directly to the round 2 real-time PCR reaction mixtures as template.

[00126] Round two - RT-PCR reaction and primers

[00127] The second round real-time PCR was designed as two separate reactions; one to detect the orfX gene, and the other to detect the mecA gene.

[00128] Round two -RT-PCR reaction and primers reaction one orfX reaction

[00129] In one example there is described a fourth primer comprising a

polynucleotide molecule of Sequence (IIII

[00130] [second orfX] (IIII

[00131] The [second orfX] comprises a polynucleotide primer that anneals to at least a portion of the orfX gene.

[00132] In a specific example, the fourth primer is Arb3.

[00133] In other example, the fourth primer is least 80% identical, such as 80%,

81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,

96%, 97%, 98%, 99%, or 100% identical to Arb3.

[00134] In one example there is described a fifth primer comprising a

polynucleotide molecule of Sequence (V),

[00135] [right side first tail] (V).

[00136] The [right side first tail] comprises a polynucleotide primer that anneals to a right side of the first tail of the first long range primer.

[00137] In a specific example, the fifth primer is SA-4.

[00138] In other example, the fifth primer is least 80% identical, such as 80%, 81 %,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SA-4.

[00139] In one example, there is described a sixth primer comprising a

polynucleotide molecule of Sequence (VI),

[00140] [first probe] (VI).

[00141] The [first probe] comprises a polynucleotide primer that anneals to a region of the orfX gene.

[00142] In a specific example, the sixth primer is SA-HEX-1.

[00143] In other example, the sixth primer is least 80% identical, such as 80%,

81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SA-HEX-1. [00144] Thus, in a specific example for the orfX reaction, amplification primer SA-4 is specific to a region in the S. aureus orfX gene, while primer Arb3 is specific to the right side tail of primer SA-3e. By targeting the tail we eliminate the possibility of amplifying from contaminating gDNA. Probe SA-HEX-1 , also specific to a region in the S. aureus orfX gene, was used to detect the PCR product specific for SA.

[00145] Round two -RT-PCR reaction and primers - reaction two - mecA reaction

[00146] In one example there is described a seventh primer comprising a polynucleotide molecule of Sequence (VII),

[00147] [UmecR/UmecRX] (VII).

[00148] The [UmecR/UmecRX] comprises a polynucleotide primer that anneals to at least a portion of [second tail] of the second long range primer and the third long range primer.

[00149] In a specific example, the seventh primer is Arb2.

[00150] In other example, the seventh primer is least 80% identical, such as 80%,

81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Arb2.

[00151] In one example there is described an eighth primer comprising a polynucleotide molecule of Sequence (VIII),

[00152] [UmecR primer] (VIII).

[00153] The [UmecR primer] comprises a polynucleotide primer that anneals to at least a portion of the mecA gene in the typical orientation

[00154] In a specific example, [UmexR peimer] is Umec-R.

[00155] In other example, the eighth primer is least 80% identical, such as 80%,

81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Umec-R.

[00156] In one example there is described a ninth primer comprising a polynucleotide molecule of Sequence (IX),

[00157] [Umec-RX primer] (IX).

[00158] The [Umec-RX primer] comprises a polynucleotide primer that anneals to at least a portion of the mecA gene in the reversed orientation.

[00159] In a specific example, [Umec-RX primer] is Umec-RX.

[00160] In other example, the ninth primer is least 80% identical, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Umec-RX. [00161] In one example, there is described a tenth primer comprising a polynucleotide molecule of Sequence (X),

[00162] [mec-FAM-2 detection probe] (X).

[00163] The [mec-FAM-2 detection probe] comprises a polynucleotide primer that anneals to at least a portion of the mecA gene.

[00164] In a specific example, [mec-FAM-2 detectionprobe] is m-eFcAM-2.

[00165] In other example, the tenth primer is least 80% identical, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to m-eFcAM-2.

[00166] In one example, there is described an eleventh primer comprising a polynucleotide molecule of Sequence (XI),

[00167] [mec-FAM-3 detection probe] (XI).

[00168] The [mec-FAM-3 detection probe] comprises a polynucleotide primer that anneals to at least a portion of the mecA gene.

[00169] In a specific example, [mec-FAM-3 detection probe] is m-eFcAM-3.

[00170] In other example, the eleventh primer is least 80% identical, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to m-eFcAM-3.

[00171] In one example, there is described a twelfth primer comprising a polynucleotide molecule of Sequence (XII),

[00172] [mec-FAM-4 detection probe] (XII).

[00173] The [mec-FAM-3 detection probe] comprises a polynucleotide primer that anneals to at least a portion of the mecA gene.

[00174] In a specific example, [mec-FAM-4 detection probe] is m-eFcAM-4.

[00175] In other example, the twelfth primer is least 80% identical, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to m-eFcAM-4.

[00176] Thus, in a specific example, for the mecA reaction, primer Arb2 is specific to the left side tail of UmecF/UmecFX, while primer UmecR is specific to the mecA gene in the typical orientation, and Umec-RX is specific to the mecA gene in the reversed orientation. Because of slight sequence variations in the mecA gene sequence, Probes mec-FAM-2 and mec-FAM-3 were both needed to detect the PCR products, and me-c FAM-4 needed for detecting the product from SCCmec X [00177] Detection of MRS A DNA in a sample

[00178] In some examples, there is described A method of detecting in a sample from a subject a methicillin-resistant Staphylococcus aureus (MRSA), DNA, comprising:

[00179] a) performing a first amplification reaction, comprising, combining said sample with a first long range primer of any one of claims 1 to 6, a second long range primer of any one of claims 7 to 1 1 , and a third long range primer of any one of claims 12 to 16, under amplification conditions to obtain a first amplification product,

[00180] b) performing a second amplification reaction, comprising, combining a portion of said first amplification product with a fourth primer of any one of claims 17 to 19, a fifth primer of any one of claims 20 to 22, and a sixth primer of any one of claims 23 to 25, under amplification conditions to detect a signal indicative of orfX, if MRSA DNA is present in said sample, and

[00181] c) performing a third amplification reaction, comprising combining a portion of said first amplification product with a seventh primer of any one of claims 26 to 28, an eighth primer of any one of claims 29 to 31 , a ninth primer of any one of claims 32 to 34, a tenth primer of any one of claims 35 to 37, an eleventh primer of any one of claims 38 to 40, and a twelfth primer of any one of claims 41 to 43, under amplification conditions to detect a signal indicative of mecA, if MRSA DNA is present in said sample.

[00182] Method of the invention are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[00183] In one example there is provided a kit comprising: a containing, and/or a first long range primer of any one of claims 1 to 6, and/or a second long range primer of any one of claims 7 to 1 1 , and/or a third long range primer of any one of claims 12 to 16, a fourth primer of any one of claims 17 to 19, and/or a fifth primer of any one of claims 20 to 22, and/or a sixth primer of any one of claims 23 to 25, a seventh primer of any one of claims 26 to 28, and/or an eighth primer of any one of claims 29 to 31 , and/or a ninth primer of any one of claims 32 to 34, and/or a tenth primer of any one of claims 35 to 37, and/or an eleventh primer of any one of claims 38 to 40, and/or a twelfth primer of any one of claims 41 to 43.

[00184] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway. [00185] EXAMPLES

[00186] Here we present a novel scheme for the direct detection of MRSA from clinical samples, which is designed to circumvent the false positive and negative issues of the currently available commercial assays. This novel 2 step assay uses a small number of primers that simultaneously target both the mecA and SA specific orfX genes, and are able to accurately detect all MRSA tested to date, including SCCmec control types l-XI.

[00187] RESULTS

[00188] A novel scheme for direct MRSA detection designed to overcome the deficits of commercially available assays. Currently available commercial MRSA detection assays rely on quantitative real-time PCR-based methods targeting the 3’ end of the orfX gene along with the right extremity of SCCmec, but not directly targeting the mecA gene. Both, however, suffer from the detection of false positives (due to SCC-like elements that lack mecA) and false negatives (due to the inability to detect new, variant or non-typeable SCCmec cassettes with the existing primers). We developed a novel MRSA detection assay scheme, designed to overcome issues present in the existing commercial assays.

[00189] Our novel assay is comprised of 2 PCR steps (Figurel) , including an initial long range (LR-) PCR reaction (Round 1), the product of which acts as the template for the second round real-time PCR reactions (Round 2). Forward and reverse primers for the initial LR- PCR reaction are located in the orfX and mecA genes, creating a PCR product that ranges in size from 2.3 kb to 42.3 kb (Tables 1 and 2). By simultaneously targeting both of these regions, the assay was designed to eliminate templates that do not contain the mecA gene, as well as anything that is not Staphylococcus aureus. Primer Umec-F targets a conserved sequence in the mecA/mecC gene, present in all SCCmec types except type X. Since the orientation of mecA in SCCmec X is reversed compared to the other types, primer Umec-FX was included to effectively detect that type, as well as any future SCCmec types with the reversed mecA orientation. Primer SA-3e, on the other hand, targets a region that is specific to the S. aureus orfX gene and is universally found in all S. aureus with available sequences. The target sequence differs significantly enough from that found in CoNS, thereby preventing amplification from CoNS orfX genes. Primer SA-3e also carries a 5’ biotin label, allowing the LR-PCR products to be captured with streptavidin coated magnetic beads, thereby concentrating them and purifying them of residual LR-PCR reaction components. Magnetic beads containing captured LR-PCR product from Round 1 are added directly to the round 2 real-time PCR reaction mixtures as template. All 3 LR-PCR primers were also designed with a 20 or 21 bp tail sequence on the 5’ end that do not match any bacterial genomes published thus far (Table 1 , underlined sequence). These right ( orfX side) and left ( mecA side) tail sequences act as templates for round 2 PCR primers, eliminating the possibility of amplification directly from any contaminating chromosomal DNA in the Round 2 reactions.

[00190] In Table 1 , FAM refers to fluorescein amidite; ZEN - a registered trademark of Integrated DNA Technologies for its specific double quencher system; HEX -Hexachlorofluorescein; ABkFQ ia a generic quencher that is available by various companies.

[00191] In some example, alternate quenches and/or fluorescent moieties may be used.

[00192] In some examples, the quencher may be ZEN, ABkFQ, and/or TAMRA.

[00193] In some examples, the fluorescent moiety may be FAM, Tet, and/or ROX. [00194] Is some examples, the primers do not include capture moieties and/or quenchers and/or a fluorescent moiety, as show in Table 1 B

[00195] Table 1 B

[00196] The second round real-time PCR was designed as 2 separate reactions; one to detect the orfX gene, and the other to detect the mecA gene. For the orfX reaction, amplification primer SA-4 is specific to a region in the S. aureus orfX gene, while primer Arb3 is specific to the right side tail of primer SA-3e. As mentioned, by targeting the tail we eliminate the possibility of amplifying from contaminating gDNA. Probe SA-HEX-1 , also specific to a region in the S. aureus orfX gene, was used to detect the PCR product specific for SA. For the mecA reaction, primer Arb2 is specific to the left side tail of UmecF/UmecFX, while primer UmecR is specific to the mecA gene in the typical orientation, and Umec-RX is specific to the mecA gene in the reversed orientation.

Because of slight sequence variations in the mecA gene sequence, Probes mec-FAM-2 andmec -FAM-3 were both needed to detect the PCR products, andmec -FAM-4 needed for detecting the product from SCCmec X.

[00197] Many layers of specificity have been built into the assay by careful selection of the target sequences for the long range primers, Round 2 primers and probes. However, due to the erroneous nature of long range PCR, whereby short PCR products can be generated from one correctly annealing and one mis-priming primer, we determined that it was essential to detect from both the mecA and orfX sides. While these mis-primed products are too few to detect on a traditional gel, they become amplified in the Round 2 reactions and create signals in one reaction or the other. In MSSA we noted that there was a positive signal from the orfX reaction, while in methicillin-resistant (MR) CoNS we noted a positive signal from the mecA reaction. In each case, however, the signal from the other reaction was negative, allowing us to discriminate between these strains and true MRSA, which are the only ones positive for both the mecA and orfX reactions.

[00198] The MRSA assay is capable of detecting all SCCmec types l-XIII. The ability of the assay to detect a wide variety of SCCmec types was assessed with 28 types or subtypes, including SCCmec l-XIII. While SCCmec XII has also been described (18), it has not been possible to obtain the strain from China so we could not test it, however, the assay should be able to detect it as well. During initial assay development, LR-PCR products from representative SCCmec types l-XIII were run on a 0.7% agarose gel to visualize them (data not shown). Anticipated bands were not seen for any SCCmec type, however, with the small number of cycles being used, this result was not unexpected. Multiple bands were seen for some types (such as in type I and X), while in other types a smear was seen (such as type II and VIII). As with the LR-PCR products, second round real-time PCR reaction products were also run on a 2% agarose gel during assay development. With the orfX reaction, the anticipated band was seen strongly at 196 bp for all representative SCCmec types, along with weaker bands that were larger in size. For the mecA reaction, multiple bands were seen for most SCCmec types, with the expected one at 213 bp being weak (or equivalent to the other ones) (data not shown). The notable exception was in SCCmec X, where the expected band at 179 bp was the only one visible. Despite the non-ideal results seen with these gels, the small number of full-length amplicons generated during LR-PCR were sufficient to act to act as templates for the second round reactions, with the product correctly detected by virtue of the probe specificity. Corresponding real-time PCR curves for each of the round 2 amplicons are shown in Figures 2a and b. When the complete set of SCCmec types was tested, all 27 types and subtypes were found to be positive for both orfX and mecA reactions and, consequently, positively identified as MRSA with our assay (Table 2). C(t) values for the orfX reaction ranged from 8.58 (type IIA) to 24.28 (type lIb), while C(t) values for the mecA reaction ranged from 12.54 (type X) to 26.29 (type lll_Hg).

[00199] Table 2. The assay detected correctly all SCC mec type prototypic strains

[00200] CND: could not determine since the full sequence is not available in NCBI

[00201] ^† NA: strain was not available to test

[00202] *mecA gene is inserted into the chromosome in the opposite orientation.

[00203] “+”: strain determined to be MRSA.

[00204] "-": Strain determined to be non-MRSA

[00205] Specificity of the assay. Because direct patient samples will contain a mixture of organisms, we needed to determine how specific the assay was, and if it would exclude cross-reactions with other species. This was accomplished using a panel of 40 non-MRSA, including 32 non-Staphylococci strains. In total there were 6 CoNS species (including 3 strains of S. epidermidis), and 31 strains from other diverse genera (including 2 species of Streptococcus) (Table 3). All of the isolates, including the 7 coagulase- negative Staphylococcal species, were negative with the orfX reaction, and all but 2 of the isolates were negative with the mecA reaction. This indicates that the assay does not cross react with other species and is very specific to MRSA. The noted exceptions were ATCC 29971 (Staphylococcus xylosus) and ATCC 35984 (Staphylococcus epidermidis), both of which are MeR-CoNS and, consequently, positive for the mecA target. As mentioned, both failed to produce a signal on the orfX side, indicating they were not S.

aureus and found to be negative for MRSA.

Table 3. Assay specificity in various species and strains of coagulase-negative staphylococci and non-staphylococcal bacteria

[00206] Sensitivity of the assay. MRSA assay sensitivity was examined with 6 representative SCCmec types, covering a range of orfX-mecA interval sizes, both mecA and mecC genes, as well as the reversed mecA of type X. Tenfold dilutions of purified gDNA were prepared and tested. Under standard conditions the limit of detection for the orfX reaction was 17 CFU/PCR for type II, 320 CFU/PCR for type III, 72.4 CFU/PCR for type IVa, 4.4x10³ CFU/PCR for type IX, 16.4 CFU/PCR for type X, and 2.8 CFU/PCR for type XI. The limit of detection for the mecA reaction was 17 CFU/PCR for type II, 3.2x10⁴ CFU/PCR for type III, 720 CFU/PCR for type IVa, 4.4x10⁴ CFU/PCR for type IX, 16.4 CFU/PCR for type X, and 2.8 CFU/PCR for type XI. As both targets need to be positive for a sample to be considered MRSA positive, the overall limit of detection for the assay is limited by the least sensitive reaction (the mecA reaction in most cases).

[00207] Table 4 depict the results of assay sensitivity in various

representative SCC mec type prototypic strains [00208] Table 4. Assay sensitivity in various representative SCC mec type prototypic strains

[00209] Previous false positive isolates were accurately classified with our

MRSA detection assay. A study validating the commercially available Xpert MRSA nasal assay had previously detected 20 MSSA isolates falsely identified as MRSA, although one of the isolates was later determined to be a true MRSA carrying SCCmec IVc (Arbefeville, 201 1). Strains Iowa 1-20 were tested with our MRSA detection assay and all 20 were found to be positive for the orfX reaction, while all but one were negative for the mecA reaction. Iowa-16 was positive for both the orfX and mecA reactions and classified as MRSA, which is in agreement with the Arbefeville study.

[00210] Assay validation with random clinical isolates. Validation of our assay was done using a collection of 218 randomly selected clinical isolates that had previously undergone extensive genetic characterization. Among them were 73 MRSA, 75 MSSA,

43 MeR-CoNS, and 27 MeS-CoNS isolates (Table 5). Of the 73 MRSA, 41 strains were previously determined to be SCCmec II, 10 were type IIIHg, 3 were type IVa, 1 was type IVb and 18 carried an unknown SCCmec type. All 73 MRSA were positive in both the mecA and orfX reactions, with the orfX reaction having a lower C(t) than the mecA reaction (approximately 10 C(t) values lower (data not shown)). All 75 MSSA had positive C(t) value for the orfX reaction, but remained negative for the mecA reaction. Similarly, all 43 MeR-CoNS had a positive C(t) value for the mecA reaction, but remained negative for the orfX reaction. All 27 MeS-CoNS were negative for both the orfX and mecA reactions. Table 5 summarizes results for the 218 clinical isolates, showing that the assay is capable of correctly identifying MRSA with 100% accuracy.

[00211]

Table 5. Assay validation in 218 random clinical isolates.

[00212] Assay applicability determined with direct clinical samples. The ability of the assay to detect MRSA directly in patient samples was assessed with duplicate swabs collected from hospitalized patients previously known to be MRSA positive, and from patients attending the sexually transmitted infection clinic in our local health region. A total of 42 samples were obtained from patients attending the sexually transmitted clinic, representing 14 patients with sampling from 3 locations for each patient. As seen in Table 6, none of the samples from the clinic were positive for MRSA using the real-time PCR assay, and no MRSA was isolated by routine clinical culture, meaning there was 100% agreement between the 2 methods.

[00213] Table 6. Assay applicability assessed with swabs from random patients attending an outpatient clinic.

[00214] Note: N, nasal; T, throat; K, perianal-perineal; V, vaginal; G, groin; + positive result (strain type was present or PCR was positive); negative result (strain type was not present or PCR was negative); clinical culture and PCR assay agree.

[00215] A total of 46 samples were obtained from hospital inpatients previously known to be MRSA positive, representing 19 patients with 2-3 locations sampled for each patient. As shown in Table 7, there was a high degree of concordance between the realtime PCR assay results and clinical culture results.

[00216] Table 7. Assay applicability assessed with swabs from hospital inpatients previously known to be MRSA positive.

[00217] Note: N, nasal; K, perianal-perineal; G, groin; Z, z-swab; W, wound; + positive result (strain type was present or PCR was positive); -, negative result (strain type was not present or PCR was negative); ¨, 5-10 colonies; 1 1 -100 colonies; ¨¨ , 101 -1000 colonies; n , clinical culture and PCR assay agree; *, clinical culture and PCR assay do not agree; -* and No MRSA*, not found on plate but recovered after overnight incubation in TSB; n*, did not agree with initial plate culture, but agreed after recovery in TSB; unk, Unknown because colonies not isolated.23 patients were found to be MRSA positive by clinical culture, and 25 MRSA positive by PCR, with the results for 84/88 (95.5%) swabs agreeing between the two methods. The PCR assay was able to detect low and high levels of MRSA on the swabs, ranging from 2 to several 100 colonies present on the plate. The results for a total of 4 swabs differed between the 2 assays. 1 swab was PCR negative, but 5-10 MRSA colonies were detected on the culture plate. 1 swab was PCR positive but plate culture negative, however, following overnight incubation of the swab in Tryptic soy broth (TSB), MRSA was detected in culture. Finally, 2 swabs were PCR positive but culture negative, and no MRSA was detected even after overnight incubation of the swabs in TSB broth. PPV/NPV SCCmec typing of the MRSA that were isolated by clinical culture revealed that the majority of them (20/27) belonged to SCCmec IVa, 1/27 to SCCmec IVc, 2/27 to SCCmec II and 2/27 to SCCmec V. Two could not be determined because they were not isolated on plate culture, leaving no strain to type. [00218] DISCUSSION

[00219] Molecular detection methods have been developed, but are hampered by the fact that clinical samples can be mixed, containing combinations of MSSA, MRSA, and MS- or MR-CoNS. Detection of the mecA gene does not necessarily indicate the presence of MRSA, as MR-CoNS could also be the source of the gene. Two FDA- approved commercially available PCR based assays have been described and are widely used to detect MRSA directly from clinical samples, including IDI-MRSA/GeneOhm MRSA (BD Diagnostics) and GeneXpert MRSA (Cepheid). In the IDI-MRSA assay, there is a SA specific primer (Xsau325) in the orfX gene, immediately upstream of attB, along with a SA specific probes which gives species specificity. The other five SCCmec specific primers (mecii574,mec iii519,mec iv51 1 , mecv 492, mecvii12) are in the J3 region of SCCmec, and are used along with Xsau325 to amplify the junction region of MRSA (19). The GeneXpert MRSA assay is similarly designed, with primers spanning the

chromosome orfX-SCCmec junction, with amplification and detection occurring in separate chambers of single-use disposable cartridge.

[00220] A study by Buchan et al. compared both the IDI-MRSA and GeneXpert MRSA to routine clinical culture for detection of MRSA in positive blood culture broths and found that they both have a relatively high sensitivity and specificity. The GeneOhm assay had a sensitivity of 99.2% (range, 95.2-100%) and specificity of 96.5 % (range, 89.2-100%) for identifying S. aureus, and a sensitivity of 94.3% (range, 87.5-100%) and specificity of 97 (range, 96.1-100%) for identifying MRSA (20). For the GeneXpert assay they noted a sensitivity of 99.6% (range, 96.4-100%) and specificity of 99.5% (range, 98- 100%) for identifying S. aureus, and a sensitivity of 981.% (range, 87.5-100%) and specificity of 99.6% (range, 98.3-100%) for identifying MRSA (20). Multiple studies, however, have found that both assays are problematic in that they produce false negatives from their inability to detect variant or new SCCmec types with the existing, and false positives from their detection of SCC-like elements that do not contain mecA primers (13-17). A study by Stamper et al found that the GeneOhm MRSA assay had a sensitivity and specificity of 89 and 91.7% respectively, with false positives resulting from retained segments of the right extremity-junction sequences in non -mecA containing SCC-like elements (17). Likewise, as study by Arbefeville et al found that the rate of false positives for the Xpert MRSA assay was 7.7%, due to mecA dropout strains with remnants of SCCmec cassettes (13). Both assays are also problematic in that they are unable to detect the newly emerging variant SCCmec types. [00221] With the goal of improving point of care detection, we developed a new real-time PCR assay for the detection of MRSA in clinical samples that circumvents the major problems encountered with commercially available assays. Our assay

simultaneously targets both the chromosomal orfX region and the mecA gene in a long range PCR step. This long-range PCR product becomes the template for a second round reaction, in which real-time PCR amplification and detection is done with primers and probes specific to both the SA orfX and mecA genes. The assay has the advantage of only needing a small number of primers to detect a large range of SCCmec types.

Because of the highly homologous nature of both the orfX and mecA genes, these limited primers are capable of detecting all SCCmec types described to date, including SCCmec XI which carries the new mecC homologue. Additional primers were added to account for the reversed orientation of the mecA gene in SCCmec type X, and would likely be effective in any future types that are described containing a reversed mecA gene. With the assay, we were able to correctly detect 27 SCCmec types and subtypes, ranging from SCCmec I to XI (Table 3) using purified DNA preparations. The high degree of assay specificity was further demonstrated by testing 40 non-MRSA isolates, including 32 non- Staphylococcal species. 100% of them were negative in the MRSA detection assay, showing that the assay is highly specific and would effectively distinguish MRSA from other bacterial special in direct clinical samples.

[00222] When applied against 218 clinical isolates that had previously undergone extensive genetic testing 100% sensitivity and specificity were noted. In all cases, MRSA were found to be positive in both the orfX and mecA reactions, while MSSA was only positive in the orfX reaction, MR-CoNS was only positive in the mecA reaction, and MS- CoNS was negative for both reactions. The cause of the positive orfX signal with MSSA, and positive mecA signal with MR-CoNS, is believed to be due non-specific primer annealing. While one primer matches perfectly, the second one mis-primes internal to the SCCmec element, producing too few products to be visualized by gel, but a strong signal upon re-amplification in the second round reactions. However, because MSSA and MR- CNS are only positive for one of the two signals, they can easily be distinguished from true MRSA, which is positive for both.

[00223] Applicability of the assay was assessed using clinical swabs from inpatients previously known to be MRSA positive, as well as from random patients at a clinic in our health region. Results indicate that the assay has a high level of sensitivity and accuracy. A total of 88 swabs from 33 patients were tested with the real-time PCR assay, and the results compared to duplicate swabs subjected to routine clinical culture. 95.5% of the swabs agreed between the 2 method, with the results for 4 swabs differing. One swab was culture positive and PCR negative, however, only 5-10 colonies were seen on the culture plate, meaning the patient had very low levels of MRSA. This could represent a false negative because the MRSA strain carried SCO mec II, which has a larger orfX-mecA size, however, the type II control strain had a low experimental limit of detection of 17 cells/reaction. It’s also possible that the discrepancy and false negative resulted from differences in sampling, since the PCR assay and plate culture were done using separate swabs, particularly with such low levels detected in the culture. At the other end of the spectrum, 3 of the swabs were PCR positive and culture negative, although one sample was found to contain MRSA following incubation of the swab in broth culture overnight. This suggests that the sensitivity of the real-time PCR assay is superior to that of culture, although as mentioned, sampling of the duplicate swabs could potentially account for the discrepancies, particularly if low levels of bacteria are present. The differences could also be due to the presence of non-viable MRSA on the swabs, unable to grow in culture, but with DNA (either within or released from the dead cells) still present on the swab. These inpatients were known to be MRSA positive, and all 3 were on antibiotic therapy prior to the samples being collected. Assuming that the 3 swabs were in fact positive for MRSA, the real-time PCR assay correctly identified 87/88 (98.9%) of the swabs, while clinical culture correctly identified 85/88 (96.6%). An interesting observation was that the real-time PCR assay was able to detect MRSA with higher sensitivity than the experimental limits of detection suggested. 14 of the 25 swabs that were positive for MRSA had <100 colonies on the culture plates, with 9 of those having <10. The majority of them were SCC mec IV (specifically IVa) , which had an experimental limit of detection of 720 CFU/reaction.

[00224] This novel MRSA detection assay has successfully overcome 2 of the greatest deficits of previously available commercial assays; namely the issue of false negatives from new or novel SCCmec types, and false positives from non -mecA containing SCC-like elements (13-17). False negatives are eliminated due to the universal nature of the targets and primers chosen for the long-range PCR. The orfX gene is highly homologous amongst S. aureus strains, and differs from that in CoNS enough to allow selection of a region that is specific to, yet present in, all SA. Similarly, the mecA gene is nearly identical in all Staphylococci, and similar enough to the mecC gene, that a common region was located. By amplifying from these universal regions, rather than targeting sequences specific to each SCCmec type, this assay has been shown to detect the majority of SCCmec types and subtypes described to date (types IVk and XII were not available) and should, be effective in detecting newly discovered types.

In fact, of the 73 MRSA clinical samples that were used to validate the assay, 18 of them were untypeable using previously described SCCmec typing assays (21 , 22). Despite carrying an unknown SCCmec type, the assay was able to accurately classify them as MRSA. The second deficit of previously described MRSA detection assays was the issue of false positives, possibly resulting from the presence of non -mecA containing SCC-like elements. Again, due to the assay’s design, and the simultaneous targeting of both mecA and orfX during the long-range PCR, these false positives have been eliminated. In a study by Arbefevill et al. 20 MSSA strains that were falsely positive in the Xpert MRSA nasal assay were tested and 19 of the 20 isolates were indeed found to be mecA negative (13). While the authors did not have a clear explanation for the false reactivity in the Xpert MRSA assay, they postulated that it could be due to the presence of SCCmec- orfX right extremity components. Screening these isolates with our assay resulted in all 20 of them being positive for the orfX target, and all but one negative for the mecA target (thereby scoring as negative). As reported by their group, one isolate was determined to be MRSA and correctly identified. SCC-like elements present in CoNS are also negative for both targets with our assay, eliminating this potential source of false positives. Of the 27 MeS-CoNS clinical isolates that were tested, 3 of them were determined to have a non -mecA SCC element in previous studies (data not shown), and all remained negative for both targets with this assay. ATCC12228 is also described as carrying a non -mecA SCC-like element, known as SCCpbp-4 (23), and was negative for both targets with our assay.

[00225] Our MRSA detection real-time PCR assay has proven to be an effective method of identifying MRSA directly in clinical samples. Due to its design, in which both the orfX and mecA genes are simultaneously targeted, false positive and false negatives observed with other clinically approved point of care detection assays are eliminated. The assay can detect all SCCmec types described to date and should be effective at detecting most future ones that arise.

[00226] MATERIALS AND METHODS

[00227] Bacterial strains and culture conditions. Bacterial strains were grown overnight on Tryptic soy agar plates at 37°C. Control strains, including SCCmec I (NCTC 10442), type II (N315), type IIIHg (85/2082), type IMA (JCSC290), type IVa (CA05) , type IVb (8/6-3P), type IVc (MR108), type IVd (JCSC4469), IVg (JCSC6673), IVh (JCSC6674), I Vi (JCSC 6668), IVj (JCSC6670), type V (WIS [WBG8318]-JCSC3624), VII (JCSC6082), IX (JCSC6943), X (JCSC6945), and XI (LGA251) were obtained from were obtained from K. Hiramatsu and T. Ito at the Juntendo University in Tokyo, Japan.

SCCmec IIA (AR14/0298), MB (AR05/0.1345), IIC (AR14/2188), IID (AR13/3635.2), HE (AR13/3330.2), IVE (AR43/3330.1), and IVF (AR43/3636.1) were obtained from D.

Coleman at the University of Dublin, Ireland. SCCmec VI (HDE288) was obtained from H. de Lencastre at The Rockefeller University, New York, USA. SCCmec Mb (05MS-150) and VIII (C10682) were recovered from patients in our local hospitals or clinics. Strains Iowa 1-20 were obtained from Dr. S. Richter, University of Iowa Health Care, Iowa, USA (13).

[00228] DNA extraction. DNA was extracted by rapid boiling method, whereby 1-5 colonies were suspended in 75 ml of sterile distilled water, then heated to 95°C for 10 minutes. After centrifugation at 13,000 rpm, supernatant was used as template in the PCR reaction. Alternatively, purified DNA was extracted using the QIAamp DNA mini kit, as per the recommended protocol, with elution in 50ml of sterile distilled water.

[00229] Primer design. Primers and probes for the MRSA detection real-time PCR assay were designed following comprehensive analysis of the orfX and mecA genes, in both coagulase-negative (CoNS) Staphylococci and Staphylococcus aureus (SA).

Primers and their sequences are listed in Table 1.

[00230] Long range PCR conditions. Round one LR-PCR uses primers SA-3e, Umec-F and Umec-FX (Table 1). DNA template was derived from either crude boiled extracts, or from Qiagen kit extraction as described. 1 ml of template DNA was added to 24 ml of reaction mixture containing 0.2 mM of each primer, 1X LA PCR buffer II (TaKaRa Bio inc), 0.4 mM of each dNTP (TaKaRa Bio inc), and 1 unit of LA Taq HS (TaKaRa Bio inc). Amplification was performed in a 2720 thermal cycler (Applied Biosystems, Foster City, CA) using 15 cycles of 98°C for 10 sec, 58°C for 15 sec, and 68°C for 20 minutes, followed by holding at 4°C.

[00231] Magnetic bead capture and PCR product washing. 10 pi of 10mg/ml streptavidin coated magnetic beads (Dynabeads M-270 streptavidin; Invitrogen) was used for each LR-PCR reaction. Beads were pooled in a 1.5 ml microcentrifuge tube and washed twice in 1 ml of 1X binding and washing buffer containing 5 mM Tris-HCL, pH7.5, 0.5 mM EDTA, and 1 M NaCI, with separation from the buffer accomplished with a DynaMag-2 (Invitrogen, Oslo, Norway). Following the washes, beads were re-suspened in 20 ml of 2X binding and washing buffer (10 mM Tris-HCL, pH7.5, 1 mM EDTA, 2 M NaCI) per reaction, and 20 ml of suspension added to each LR-PCR tube. Tubes were incubated at room temperature for 45 minutes, with gentle mixing by inversion every 5 minutes. Following binding, magnetic beads were washed 2 times with 100 ml of 1X binding and washing buffer, with mixing and separation accomplished using a DynaMag- 96 Side (Invitrogen, Oslo, Norway). The samples was re-suspended in 100 pi of 10 mM Tris buffer, then 50 ml transferred to each of 2 real-time PCR tubes. Using the magnet, buffer was removed from the tubes, leaving the DNA bound beads as template for the second round real-time PCR.

[00232] Round 2 real-time PCR conditions. Round 2 real-time PCR was set up as 2 reactions for each sample. Detection of the Staphylococcus aureus specific orfX gene was accomplished with the first reaction, using primers Arb3 and SA-4. The 5’ PrimeTime qPCR 5’ nuclease probe, SA-HEX-1 , was used for PCR product detection (Integreated DNA technologies, Skokie, Illinois) (Table 1). Detection of the mecA gene was accomplished with the second reaction, using primers Arb2 along with primers Umec-R and Umec-RX. The 5’ PrimeTime qPCR 5’ nuclease probes me-cFAM-2, mec- FAM-3 and mec-FAM-4 were used for PCR product detection in this reaction (Integreated DNA technologies, Skokie, Illinois) (Table 1). The reactions were made using 1 mM of each of the appropriate primers, along with 0.25 mM of each corresponding probe and 1X PrimeTime gene expression master mix (Integreated DNA technologies, Skokie, Illinois), in a final volume of 10 ml. 10 ml of the orfX mix was added to one of the tubes containing washed beads, while 10 ml of the mecA mix was added to the second tube of washed beads. Both reactions are run simultaneously in the same real-time PCR thermocycler with the following conditions: an initial incubation at 95°C for 2 minutes was followed by 45 cycles of 95°C for 10 seconds and 65°C for 20 seconds, with at HEX read for the tubes containing the orfX mix, or a FAM read for tubes containing the mecA mix.

Reactions were considered positive when the C(t) value was between 1-38, and negative if there was no C(t) value, or if it was greater than 38.

[00233] Assay sensitivity, specificity and validation. The ability of our MRSA detection assay to detect a large variety of SCCmec types was assessed using SCCmec control strains, including types I, II, IIA, MB, IIC, IID, HE, lIb, IIIHg, Ilia, IVa, IVb, IVc , IVd, IVE, IVF, IVg, IVh, IVi, IVj, V, VI, VII, VIII, IX, X and XI. Assay cross-reactivity was determined using a collection of non MRSA strains, including JCSC5402 (M.

caseolyticus), ATCC 29970 (Staphylococcus haemolyticus), ATCC 12333

(Staphylococcus epidermidis), ATCC 27851 (Staphylococcus simulans), ATCC 35984 (Staphylococcus epidermidis), ATCC 29971 (Staphylococcus xylosus), ATCC 15305 (Staphylococcus saprophyticus), ATCC 29060 (Staphylococcus sciuri), ATCC 12228 (Staphylococcus epidermidis), OP50 (Escherichia coli), ATCC 14028 (Salmonella typhymurium), ATCC 31426 (Neisseria gonorrhoeae), ATCC 4698 (Micrococcus luteus), ATCC 49143 (Moraxella catarrhalis), ATCC 51331 (Stenotrophomonas maltophilia),

ATCC 1 1778 (Bacillus cereus), ATCC 8750 (Alcaligenes faecalis subsp. faecalis), ATCC 15947 (Edwardsiella tarda), ATCC 25931 (Shigella sonnei), ATCC 35657 (Klebsiella pneumonia), ATCC 15468 (Aeromonas caviae), ATCC 1 1454 (Lactococcus lactis), ATCC 19615 (Streptococcus pyogenes), ATCC 191 15 (Listeria monocytogenes), ATCC 43862 (Serratia marcescens), ATCC 43864 (Citrobacter freundii), ATCC 23216 (Leclercia adecarboxylata), ATCC 49129 (Ralstonia pickettii), ATCC 9340 (Bordetella pertussis), ATCC 33152 (Legionella pneumophilia), ATCC 14468 (Mycobacterium smegmatis),

ATCC 10137 (Streptomyces griseus), ATCC 1 1913 (Corynebacterium diphtheria), ATCC 19606 (Acinetobacter baumannii), ATCC 17802 (Vibrio parahaemolyticus), ATCC 186 (Kocuria rosea), ATCC 49136 (Streptococcus pneumonia), ATCC 19414 (Erysipelothrix rhusiopathiae), ATCC 43534 (Oligella ureolytica), and ATCC 6056 (Enterococcus durans). 1 ml of Qiagen purified DNA was used as template.

[00234] To determine assay sensitivity, the correlation of 1.0 McFarland standard being equal to 3x108 CFU/ml was initially used. Control strains carrying SCCmec II, III, IVa, IX, X and XI were suspended in saline to a concentration of 1.0 Mcfarland standard. Exact colony counts were further confirmed by plating dilutions of the suspension on TSA plates, and growing overnight at 37°C. 1 ml of suspension was pelleted and the DNA extracted following standard procedures using the QIAamp DNA mini kit, and eluted with 50ml of sdH20. DNA concentration of the extract was determined with a Qubit 3 fluorometer using the Qubit dsDNA BR kit (Invitrogen). 10 fold serial dilution of the DNA were subsequently used to assess assay sensitivity.

[00235] The ability of the assay to correctly classify isolates falsely identified as MRSA by the Xpert MRSA nasal assay (13) was determined. 1 ml of boiled DNA from 20 MSSA strains (Iowa 1-20) was tested. The assay was subsequently validated using a collection of strains that were obtained from random clinical samples that had been extensively characterized molecularly (24, 25). DNA was extracted using the boiling method and 1 ml used in the assay.

[00236] Clinical applicability using direct patient swabs. The ability of the assay to detect MRSA directly in patient samples was assessed with 88 swabs collected from hospitalized patients previously known to be MRSA positive or randomly from patients attending the sexually transmitted infection clinic in our local health region. Swabs were collected from multiple sites on each patient (nasal (N), throat (T), perianal-perineal (K), groin (G), Z-swab (Z), wound (W), axilla (A), or vaginal (V) swab), and from each site duplicate swabs were obtained. One swab was subjected to routine clinical culture and identification, followed by genetic characterization of each isolate with a PCR multiplex assay capable of distinguish CoNS from SA and methicillin-resistant vs methicillin-sensitive isolates (24). Isolates were also subjected to SCCmec typing (22) as previously described. DNA was isolated from the second swab using the QIAmp DNA minikit (Qiagen Inc., Toronto, Ontario, Canada) following protocol D for isolation of genomic DNA from grampositive bacteria, using 200 mg/ml of lysostaphin. The entire swab was deposited into the microcentrifuge tube for extraction purposes, and DNA was eluted from the column with 50 ml of sterile water. 1 ml of the eluent was used as the template in real-time PCR assay. When PCR was found to be positive and clinical culture negative, the swab used for clinical culture was placed into 30 mis of Tryptic soy broth and grown overnight at 37 °C. 50 ml of the overnight culture was transferred to a blood plate and subjected to clinical identification and molecular typing, as described above.

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[00264] The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[00265] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00266] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A first long range primer comprising a polynucleotide molecule of Sequence (I), [capture moiety]-[first tail]-[orfX] (I).

2. The first long range primer of claim 1 , wherein said [capture moiety] comprises a polynucleotide that is labelled with biotin, beads coated/labelled with biotin, other fluorescent labels, or tags.

3. The first long range primer of claim 1 or 2, wherein said [first tail] comprises a polynucleotide sequence that does not match a known bacterial genome.

4. The first long range primer of any one of claims 1 to 3, wherein said [first tail] is 20-35 base pairs long, or 20 base pairs long or 21 base pairs long.

5. The first long range primer of any one of claims 1 to 4, wherein said [orfX] comprises a polynucleotide that anneals to at least of a portion of the orfX gene.

6. The first long range primer of any one of claims 1 to 5, wherein [capture moiety]- [first tail]-[orfX] is SA-3e (SEQ ID NO: 1).

7. A first long range primer comprising a polynucleotide molecule of Sequence (la), [orfX] (la).

8. The first long range primer of claim 7, wherein said [first tail] comprises a polynucleotide sequence that does not match a known bacterial genome.

9. The first long range primer of claim 7 or 8, wherein said [first tail] is 20 - 35 base pairs long, or 20 base pairs long or 21 base pairs long.

10. The first long range primer of any one of claims 7 to 9, wherein said [orfX] comprises a polynucleotide that anneals to at least of a portion of the orfX gene.

1 1. The first long range primer of any one of claims 7 to 10, wherein [first tail]-[orfX] is SA-3e MOD (SEQ ID NO: 13).

12. A second long range primer comprising a polynucleotide molecule of Sequence (II),

[second tail]-[mecA] (II).

13. The second long range primer of claim 12, wherein said [second tail] comprises a polynucleotide sequence that does not match a known bacterial genome.

14. The second long range primer of claim 12 or 13, wherein said [second tail] is 20- 35 base pairs long, or 20 base pairs long or 21 base pairs long.

15. The second long primer any one of claims 12 to 14, wherein said [mecA] comprises a polynucleotide that anneals to at least portion of the meAc gene.

16. The second long primer of any one of claims 12 to 15, wherein [second tail]- [mecA] is Umec-F (SEQ ID NO: 2).

17. A third long range primer comprising a polynucleotide molecule of Sequence (III), [second tail]-[reversed Umec] (III).

18. The third long range primer of claim 17, wherein said [second tail] comprises a polynucleotide sequence that does not match a known bacterial genome.

19. The third long range primer of claim 17 or 18, wherein said [second tail] is 20-35 base pairs long, or 20 base pairs long or 21 base pairs long.

20. The third long range primer of any one of claims 17 to 19, wherein said [reversed Umec] comprises a polynucleotide primer that anneals to at least a portion of reversed Umec of SCCmecX.

21. The third long range primer of any one of claims 17 to 19, wherein the [second tail]-[reversed Umec] is Umec-FX is (SEQ ID NO: 3).

22. A fourth primer comprising a polynucleotide molecule of Sequence (IV),

[second orfX] (IV).

23. The fourth primer of claim 22, wherein [second orfX] comprises a polynucleotide primer that anneals to at least a portion of the orfX gene.

24. The fourth primer of claim 22 or 23, wherein [second orfX] is Arb3 (SEQ ID NO: 4).

25. A fifth primer comprising a polynucleotide molecule of Sequence (V),

[right side first tail] (V).

26. The fifth primer of claims 25, [right side first tail] comprises a polynucleotide primer that anneals to a right side of the first tail of the first long range primer.

27. The fifth primer of claim 25 or 26, wherein [right side first tail] is SA-4 (SEQ ID NO:

5).

28. A sixth primer comprising a polynucleotide molecule of Sequence (VI),

[first probe] (VI).

29. The sixth primer of claim 28, wherein [first probe] comprises a polynucleotide primer that anneals to a region of the orfX gene.

30. The sixth primer of claim 28 or 29, wherein [first probe] comprises at least one quencher and/or at least on fluorescent molecule.

31. The sixth primer of claims 30, wherein the at least one quencher is ZEN, ABkFQ, and/or TAM RA.

32. The sixth primer of claim 30 or 31 , wherein the at least fluorescent moiety is Fam, Tet, and/or ROX.

33. The sixth primer of claim 28 or 32, wherein [first probe] is SA-HEX-1 (SEQ ID NO:

6) or SA-HEX-1 MOD (SEQ ID NO: 14).

34. A seventh primer comprising a polynucleotide molecule of Sequence (VII),

[UmecF/UmecFX] (VII).

35. The seventh primer of claim 34, wherein [UmecF/UmecFX] comprises a polynucleotide primer that anneals to at least a portion of [second tail] of the second long range primer and the third long range primer.

36. The seventh primer of claim 34 or 35, wherein [UmecF/UmecFX] is Arb2 (SEQ ID NO: 7).

37. An eighth primer comprising a polynucleotide molecule of Sequence (VIII),

[UmecR primer] (VIII).

38. The eighth primer of claim 37, wherein [UmecR primer] comprises a polynucleotide primer that anneals to at least a portion of the meAc gene in the typical orientation.

39. The eighth primer of claim 37, wherein [UmexR primer] is Umec-R (SEQ ID NO: 8).

40. A ninth primer comprising a polynucleotide molecule of Sequence (IX),

[Umec-RX primer] (IX).

41. The ninth primer of claim 40, wherein [Umec-RX primer] comprises a polynucleotide primer that anneals to at least a portion of the meAc gene in the reversed orientation.

42. The ninth primer of claim 40 or 41 , wherein [Umec-RX primer] is Umec-RX (SEQ ID NO: 9).

43. A tenth primer comprising a polynucleotide molecule of Sequence (X),

[mec-FAM-2 detection probe] (X).

44. The tenth primer of claim 43, wherein [mec-FAM-2 detection probe] comprises a polynucleotide primer that anneals to at least a portion of the meAc gene.

45. The tenth primer of claim 43 or 44, wherein [mec-FMA-2 detection probe] comprises at least one quencher and/or at least on fluorescent molecule.

46. The tenth primer of claim 45, wherein the at least one quencher is ZEN, ABkFQ, and/or TAM RA.

47. The tenth primer of claim 45 or 46, wherein the least one fluorescent moiety is FAM, Tet, and/or ROX.

48. The tenth primer of any one of claims 43 to 47, wherein [mec-FAM-2 detection probe] is mec-FAM-2 (SEQ ID NO: 10) or [mec-MOD-2] (SEQ ID NO: 15).

49. An eleventh primer comprising a polynucleotide molecule of Sequence (XI),

[mec-FAM-3 detection probe] (XI).

50. The eleventh primer of claim 49, wherein [mec-FAM-3 detection primer] comprises a polynucleotide primer that anneals to at least a portion of the mecA gene.

51. The eleventh primer of claim 49 or 50, wherein [mec-FAM-3 detection primer] comprises at least one quencher and/or at least one fluorescent molecule.

52. The eleventh primer of claim 51 , wherein the at least one quencher is ZEN, ABkFQ, and/or TAMRA.

53. The eleventh primer of claim 51 or 52, wherein the at least one fluorescent moiety is FAM, Tet, and/or ROX.

54. The eleventh primer of any one of claims 49 to 53, wherein [mec-FAM-3 detection probe] is mec-FAM-3 (SEQ ID NO: 1 1) or [mec-MOD-3] (SEQ ID NO: 16).

55. A twelfth primer comprising a polynucleotide molecule of Sequence (XII),

[mec-FAM-4 detection probe] (XII).

56. The twelfth primer of claim 55, wherein [mec-FAM-3 detection primer] comprises a polynucleotide primer that anneals to at least a portion of the mecA gene.

57. The twelfth primer of claim 55 or 56, wherein [mec-FAM-3 detection primer] comprises at least one quencher and/or at least on fluorescent molecule.

58. The twelfth primer of claim 57, wherein the at least one quencher is ZEN, ABkFQ, and/or TAM RA.

59. The twelfth primer of claim 57 or 58, wherein wherein the least one fluorescent moiety is FAM, Tet, and/or ROX.

60. The twelfth primer of any one of claims 55 to 59, wherein [mec-FAM-4 detection probe] is mec-FAM-4 (SEQ ID NO: 12) or [mec-MOD-4] (SEQ ID NO: 17).

61. A method of detecting in a sample from a subject a methicillin-resistant Staphylococcus aureus (MRSA), DNA, comprising:

a) performing a first amplification reaction, comprising, combining said sample with a first long range primer of any one of claims 1 to 1 1 , a second long range primer of any one of claims 12 to 16, and a third long range primer of any one of claims 17 to 21 , under amplification conditions to obtain a first amplification product,

b) performing a second amplification reaction, comprising, combining a portion of said first amplification product with a fourth primer of any one of claims 22 to 24, a fifth primer of any one of claims 25 to 27, and a sixth primer of any one of claims 28 to 33, under amplification conditions to detect a signal indicative of orfX, if MRSA DNA is present in said sample, and

c) performing a third amplification reaction, comprising combining a portion of said first amplification product with a seventh primer of any one of claims 34 to 36, an eighth primer of any one of claims 37 to 39, a ninth primer of any one of claims 40 to 42, a tenth primer of any one of claims 43 to 48, an eleventh primer of any one of claims 49 to 54, and a twelfth primer of any one of claims 55 to 60, under amplification conditions to detect a signal indicative of mecA, if MRSA DNA is present in said sample.

62. The method of claim 61 , further comprising a step of enriching said amplification product of step a) prior to step b) and step c).

63. The method of claim 61 or 62, wherein said subject is a human.

64. A kit comprising: a containing, and/or a first long range primer of any one of claims 1 to 1 1 , and/or a second long range primer of any one of claims 12 to 16, and/or a third long range primer of any one of claims 17 to 21 , a fourth primer of any one of claims 22 to 24, and/or a fifth primer of any one of claims 25 to 27, and/or a sixth primer of any one of claims 28 to 33, a seventh primer of any one of claims 34 to 36, and/or an eighth primer of any one of claims 37 to 39, and/or a ninth primer of any one of claims 40 to 42, and/or a tenth primer of any one of claims 43 to 48, and/or an eleventh primer of any one of claims 49 to 54, and/or a twelfth primer of any one of claims 55 to 60.