Compositions and methods for detecting methicillin-resistant Staphylococcus aureus

Abstract

A method for rapid detection of the presence or absence of mecA and / or mecC-containing Staphylococcus aureus (mecA / mecC-MRSA) in a biological or non-biological sample is described. The method can include performing amplification, hybridization, and detection steps. Additionally, primers, probes targeting mecA-MRSA and mecC-MRSA genes, and kits designed for the detection of mecA / mecC-MRSA are provided.

Description

Technical Field

[0001] Field of the Invention The present disclosure relates to the field of bacterial diagnostics, and more particularly to the detection of methicillin-resistant Staphylococcus aureus (MRSA) containing mecA and mecC nucleic acid sequences.

Background Art

[0002] Background of the Invention Staphylococcus aureus (“Staphylococcus aureus” or “SA”) is a facultative anaerobic Gram-positive bacterium, and its natural reservoirs include human skin and nose, and it can also inhabit wound sites. Most people carrying Staphylococcus aureus do not show signs of infection. However, Staphylococcus aureus can become invasive when normal barriers are breached and can cause infections in the body. Staphylococcus aureus can cause a number of diseases ranging from minor skin infections such as boils, abscesses, and pustules to major diseases such as pneumonia, meningitis, and sepsis. Tissues other than the skin and nose can become infected when barriers, such as the inner layer of the skin or mucosa, are breached, which results in furuncles and carbuncles. Staphylococcus aureus infections can spread among people through skin contact with an infected person or contact with objects used by an infected person.

[0003] Staphylococcus aureus has a remarkable ability to develop resistance to major antibiotics including penicillin (methicillin, oxacillin, cloxacillin, and flucloxacillin), thus earning the label of “superbug”. Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that has become resistant to penicillin and is the cause of several human infections that are difficult to treat. MRSA is also known as oxacillin-resistant Staphylococcus aureus (ORSA) and multi-drug resistant Staphylococcus aureus, while non-methicillin-resistant strains of Staphylococcus aureus may also be called methicillin-sensitive Staphylococcus aureus (MSSA).

[0004] The gene mecA, which is required for methicillin resistance in Staphylococcus aureus, encodes a low-affinity penicillin-binding protein 2a (PBP2a) (Niemeyer et al., J. Bacteriol., (1996), 178(18):5464-5471). A novel variant of mecA, newly named mecC, has recently been identified in Staphylococcus aureus isolates from both humans and animals (Harrison et al., Antimicrob. Agents Chemother., (2013), 57(3):1524-1528). This homolog shares 70% nucleotide identity with the mecA gene, and its presence causes a diagnostic problem in that it may be misdiagnosed as methicillin-susceptible Staphylococcus aureus (Paterson et al., Trends Microbiol., (2014), 22(1):42-47). Therefore, there is a need in the art for a rapid and reliable method for specifically detecting both mecA-containing MRSA and mecC-containing MRSA in a highly sensitive manner. LGA251 ). SUMMARY OF THE INVENTION

[0005] Summary of the Invention Certain embodiments of the present disclosure relate to a method for the rapid detection of the presence or absence of mecA or mecC-containing Staphylococcus aureus (mecA / mecC-MRSA) in a biological or non-biological sample, e.g., multiplex detection of mecA / mecC-MRSA by real-time polymerase chain reaction in a single test tube. Embodiments include a method for detecting mecA / mecC-MRSA that includes performing at least one cycling step that may include an amplification step and a hybridization step. Further, embodiments include primers, probes, and kits designed for the detection of mecA / mecC-MRSA in a single test tube. The detection method is designed to target the mecA gene or the mecC gene, thereby enabling the detection of mecA / mecC-MRSA in a single test.

[0006] In one aspect, a method for detecting mecA and / or mecC-containing Staphylococcus aureus in a sample, the method comprising performing an amplification step of contacting the sample with a pair of mecA-MRSA primers and / or a pair of mecC-MRSA primers to produce an amplification product if mecA and / or mecC-MRSA is present in the sample; performing a hybridization step of contacting the amplification product with one or more detectable mecA-MRSA probes and / or one or more detectable mecC-MRSA probes; and a step of detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of mecA and / or mecC-MRSA in the sample and the absence of the amplification product indicates the absence of mecA and / or mecC-MRSA. As used herein, a pair of mecA-MRSA primers can or may consist of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 1 or its complement, and a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 2 or its complement; and / or a pair of mecC-MRSA primers can or may consist of a forward primer comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3 or 4 or its complement, and a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 5 or its complement. Further, one or more detectable mecA-MRSA probes can or may consist of the nucleic acid sequence of SEQ ID NO: 6 or its complement, and / or one or more detectable mecC-MRSA probes can or may consist of the sequence of SEQ ID NO: 7 or its complement.

[0007] In one aspect, a primer set for amplification of the mecA / mecC-MRSA gene target comprises the nucleic acid sequences of SEQ ID NOs: 1, 2, 3, 4 and 5 or their complements, and a detectable probe for detection of the mecA / mecC-MRSA amplification product comprises the nucleic acid sequences of SEQ ID NOs: 6 and 7 or their complements.

[0008] Another aspect provides an oligonucleotide comprising or consisting of a nucleic acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7 or a complement thereof, the oligonucleotide having 100 or fewer nucleotides. In another aspect, the disclosure provides an oligonucleotide comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, or 95%) with one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, or a complement thereof, the oligonucleotide having 100 or fewer nucleotides. Generally, these oligonucleotides can be primer nucleic acids, probe nucleic acids, etc. in these embodiments. In certain of these embodiments, the oligonucleotide has 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, etc.). In some embodiments, the oligonucleotide comprises at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to an unmodified nucleotide. Optionally, the oligonucleotide comprises at least one label and / or at least one quencher moiety. In some embodiments, the oligonucleotide comprises at least one conservatively modified variant. A "conservatively modified variant" or simply "conservative variant" of a particular nucleic acid sequence refers to a nucleic acid that encodes the same or substantially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, has a substantially the same sequence. One of ordinary skill in the art will recognize that individual substitutions, deletions, or additions that change, add, or delete a single amino acid or a small number of amino acids (typically less than 5%, more typically less than 4%, 2%, or 1%) in the encoded sequence are "conservative modification variants" where the modification results in a deletion of an amino acid, an addition of an amino acid, or a substitution of an amino acid with a chemically similar amino acid.

[0009] In one aspect, amplification may use a polymerase enzyme having 5’→3’ nuclease activity. Thus, the first and second fluorescent moieties may be within 8 nucleotides of each other along the length of the probe. In another aspect, the mecA-MRSA and mecC-MRSA probes contain nucleic acid sequences that allow for secondary structure formation. Such secondary structure formation generally results in spatial proximity between the first and second fluorescent moieties. According to the method, the second fluorescent moiety on the probe may be a quencher.

[0010] The present disclosure provides a method for detecting the presence or absence of mecA / mecC-MRSA in a biological sample from an individual. Such methods generally include performing at least one cycling step that includes an amplification step and a dye binding step. Typically, the amplification step includes contacting the sample with a plurality of pairs of mecA-MRSA primers and mecC-MRSA primers to produce one or more mecA / mecC-MRSA amplification products if mecA / mecC-MRSA nucleic acid molecules are present in the sample, and the dye binding step includes contacting the mecA / mecC-MRSA amplification products with a double-stranded DNA binding dye. Such methods also include detecting the presence or absence of binding of the double-stranded DNA binding dye to the amplification products, where the presence of binding indicates the presence of mecA / mecC-MRSA in the sample and the absence of binding indicates the absence of mecC-MRSA in the sample. A representative double-stranded DNA binding dye is ethidium bromide. Additionally, such methods may also include measuring the melting temperature between the mecA / mecC-MRSA amplification products and the double-stranded DNA binding dye, and the melting temperature confirms the presence or absence of mecC-MRSA.

[0011] In a further aspect, a kit is provided for detecting one or more nucleic acids of mecA and / or mecC-MRSA. The kit may include multiple sets of mecA-MRSA and / or mecC-MRSA primers specific for amplification of the mecA gene target and / or the mecC gene target; and one or more detectable mecA-MRSA and / or mecC-MRSA probes specific for detection of the mecA / mecC-MRSA amplification product. In one aspect, the kit may include a probe already labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, or may include a fluorescent moiety for labeling the probe. The kit may also further include nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase. The kit may also include an accompanying document, as well as instructions for using the primers, probes, and fluorescent dye moieties for detecting the presence or absence of mecA / mecC-MRSA in a sample. As used herein, a pair of mecA-MRSA primers may include or consist of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 1 or its complement, and a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 2 or its complement; and / or a pair of mecC-MRSA primers may include or consist of a forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3 or 4 or its complement, and a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 5 or its complement. Further, one or more detectable mecA-MRSA probes may include or consist of the nucleic acid sequence of SEQ ID NO: 6 or its complement, and / or one or more detectable mecC-MRSA probes may include or consist of the sequence of SEQ ID NO: 7 or its complement.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0013] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and from the detailed description of the embodiments, as well as from the claims.

Brief Description of the Drawings

[0014]

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Embodiments for Carrying Out the Invention

[0015] Detailed Description of the Invention Diagnosis of MRSA infection by nucleic acid amplification provides a method for rapidly and accurately detecting bacterial infections. A real-time assay for detecting mecA / mecC-MRSA in a sample is described herein. Primers and probes for detecting mecA / mecC-MRSA are provided, and products or kits containing such primers and probes are also provided. The increased sensitivity of real-time PCR for the detection of mecA / mecC-MRSA compared to other methods, as well as the improved features of real-time PCR including sample containment and real-time detection of amplification products, enable the implementation of this technology for the routine diagnosis of mecA / mecC-MRSA infections in clinical laboratories.

[0016] Both the methicillin resistance gene mecA and its homolog mecC encode a modified methicillin-resistant penicillin-binding protein (PBP2a or PBP2’) that is a penicillin-binding protein with reduced affinity for the β-lactam ring (the primary active site of β-lactam antibiotics such as penicillins, cephalosporins, and carbapenems) (Guignard et al., 2005, Curr Opin Pharmacol 5(5):479-89), which is not present in sensitive strains and is thought to have been obtained from distantly related species. Both mecA and mecC are carried on the staphylococcal chromosomal cassette mec (SCCmec), a mobile genetic element of MRSA strains. SCC elements are also present in sensitive Staphylococcus aureus, but do not carry the mecA gene or the mecC gene, or carry a non-functional mecA gene or mecC gene. Such strains may have the same right-hand junction and can therefore cause false positive results.

[0017] Detection of MRSA from nasal specimens by detecting the mecA or mecC gene and Staphylococcus aureus-specific genes is associated with a decreased positive predictive value (PPV) due to the presence of both various amounts of non-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci (MRCoNS). These combinations cannot be distinguished from MRSA because both targets are present. Depending on the prevalence of MRSA, this situation results in false-positive results of up to 30%. For better PPV, the selected target needs to be specific to MRSA. The only target currently known is the staphylococcal cassette chromosome (SCCmec), which amplifies the transposon integration site of genetic factors with the mecC gene.

[0018] The SCCmec, an SCC element of MRSA (carrying the functional mecA or mecC gene), is a transposon of highly variable length (16 kb - 67 kb) integrated into the 3' portion of open reading frame X from Staphylococcus aureus (orfX) containing the mecC gene. OrfX has no defined function in Staphylococcus aureus and is specific to Staphylococcus aureus. The integration of SCCmec creates a signature specific to MRSA.

[0019] The SCCmec element has two essential components: the ccr gene complex (ccr) and the mec gene complex (mec). The ccr gene complex consists of the ccr gene and surrounding open reading frames (ORFs), and the mec gene complex consists of the mecC gene, regulatory genes, and insertion sequences upstream or downstream of mecC.

[0020] Classification of MRSA can be based on the various genotypes of MRSA. One target for genotype-based MRSA detection and classification can be the right-end junction (RE) of SCCmec. Thus, this method of MRSA typing is called RE (right end of SCCmec) typing. This typing method utilizes the polymorphism at the right end of SCCmec DNA adjacent to the integration site among various types of SCCmec.

[0021] The detection of mecA / mecC-containing Staphylococcus aureus (mecC-MRSA) utilizes a strategy that produces amplicons at the RE junction between the orfX gene of Staphylococcus aureus and the SCCmec that harbors the mecA gene or mecC gene conferring resistance to methicillin. To achieve this, one primer is anchored to a highly conserved region of the orfX gene of Staphylococcus aureus (orfX primer), and the second primer is placed within the non-conserved RE junction of SCCmec (RE primer). The amplicons obtained from the two primers span a portion of the orfX gene and a portion of SCCmec. Due to the non-homologous nature of SCCmec at the RE junction, several different RE primers are required to achieve a broad range of most unique MRSA strains. This type of identification and detection of mecA / mecC-MRSA has been described by several groups, for example, U.S. Patent Nos. 7,449,289 and 7,838,221 by Huletsky et al.; U.S. Patent No. 8,535,888 by Aichinger et al.; U.S. Patent Nos. 9,920,381 and 10,190,178 by Johnson et al., each of which is incorporated herein by reference in its entirety. However, as described above, this strategy can cause false positive results if the mecA gene or mecC gene is defective (completely or partially) or non-functional.

[0022] The disclosed method may include performing at least one cycling step that includes amplifying one or more portions of a mecA / mecC-MRSA nucleic acid molecule gene target from a sample using one or more pairs of mecA-MRSA and / or mecC-MRSA primers. As used herein, a "mecA-MRSA primer" or "mecC-MRSA primer" refers to an oligonucleotide primer that specifically anneals to a nucleic acid sequence encoding mecA or mecC in MRSA within the SCCmec cassette and initiates DNA synthesis therefrom under appropriate conditions. Each of the mecA-MRSA or mecC-MRSA primers under consideration anneals to a target within or adjacent to each mecA / mecC-MRSA target nucleic acid molecule such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. One or more mecA / mecC-MRSA amplification products are produced provided that one or more mecA / mecC nucleic acids are present in the sample, and thus the presence of one or more mecA / mecC-MRSA amplification products indicates the presence of mecA / mecC-MRSA in the sample. The amplification products will include nucleic acid sequences complementary to one or more detectable probes for mecA-MRSA or mecC-MRSA. Each cycling step includes an amplification step, a hybridization step, and a detection step, and the sample is contacted with one or more detectable probes for mecA-MRSA or mecC-MRSA to detect the presence or absence of mecA / mecC-MRSA in the sample.

[0023] As used herein, the term "amplify" refers to the process of synthesizing nucleic acid molecules complementary to one or both strands of a template nucleic acid molecule (e.g., mecA or mecC). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing a primer to the template nucleic acid at a temperature below the melting temperature of the primer, and enzymatically extending from the primer to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and an appropriate buffer and / or cofactor (e.g., MgCl2 and / or KCl) for optimal activity of the polymerase enzyme.

[0024] As used herein, the term "primer" is known to those of skill in the art and refers to an oligomeric compound, primarily an oligonucleotide, as well as a modified oligonucleotide that can "prime" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group, whereby deoxynucleoside triphosphates are used and a 3'→5' phosphodiester bond is established, by which a template-dependent DNA polymerase can further "nucleotide" bind such that pyrophosphate is released. Thus, there is likely no fundamental difference between "primer," "oligonucleotide," or "probe" except perhaps the intended function.

[0025] The term "hybridize" refers to the annealing of one or more probes to an amplification product. Hybridization conditions typically include a temperature lower than the melting temperature of the probe but high enough to avoid non-specific hybridization of the probe.

[0026] The term "5'→3' nuclease activity" typically refers to the activity of a nucleic acid polymerase associated with nucleic acid strand synthesis, whereby nucleotides are removed from the 5' end of a nucleic acid strand.

[0027] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, meaning that it does not irreversibly denature when exposed to high temperatures for the time necessary to effect denaturation of double-stranded template nucleic acid and catalyzes the formation of primer extension products complementary to the template. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, non-thermostable polymerases can also be used in PCR assays if the enzyme is replenished.

[0028] The term "its complement" refers to a nucleic acid that is the same length as a given nucleic acid and is exactly complementary.

[0029] The terms "extension" or "elongation" as used with respect to a nucleic acid refer to the case where additional nucleotides (or other similar molecules) are incorporated into the nucleic acid. For example, a nucleic acid is optionally extended by nucleotides that incorporate a biocatalyst such as a polymerase that adds nucleotides to the 3' end of the nucleic acid.

[0030] The terms "identical" or "percent identity" in the context of two or more nucleic acid sequences refer to two or more sequences or subsequences that are identical or have the same percentage of nucleotide identities when compared or aligned for maximum correspondence, e.g., using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST programs, e.g., Altschul et al. (1990) "Basic local alignment search tool" J. Mol. Biol. 215:403-410, Gish et al. (1993) "Identification of protein coding regions by database similarity search" Nature Genet. 3:266-272, Madden et al. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141, Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" Nucleic Acids Res. 25:3389-3402, and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation" Genome Res. 7:649-656, which are hereby incorporated by reference.

[0031] Regarding an oligonucleotide, a "modified nucleotide" refers to a change in which at least one nucleotide of the oligonucleotide sequence is replaced by a different nucleotide that provides a desired property to the oligonucleotide. Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, for example, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propylgylamino-dC, C5-propylgylamino-dU, C7-propylgylamino-dA, C7-propylgylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudodU, nitropyrrole, nitroindole, 2'-O-methylriboU, 2'-O-methylriboC, N4-ethyl-dC, N-methyl-dA, and the like. Many other modified nucleotides that can be substituted in oligonucleotides are referred to herein or are known in the art. In certain embodiments, the modified nucleotide substitution modifies the melting temperature (Tm) of the oligonucleotide as compared to the melting temperature of the corresponding unmodified oligonucleotide. Further, in some embodiments, certain modified nucleotide substitutions can, for example, reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation, etc.) and increase the yield of the intended target amplicon. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference.

[0032] mecA- or mecC-containing Staphylococcus aureus (mecA / mecC-MRSA) The present disclosure provides a method for detecting mecA / mecC-MRSA, for example, by amplifying a part of the mecA or mecC nucleic acid sequence. Nucleic acid sequences of SCCmec of various subtypes of mecA / mecC-MRSA are available (for example, GenBank accession number AY786579 for mecA and GenBank accession number FR823292 for mecC). Specifically, primers and probes for amplifying and detecting mecA / mecC-MRSA nucleic acid molecular targets are provided by embodiments of the present disclosure.

[0033] For the detection of mecC-MRSA, primers and probes for amplifying the mecA-MRSA gene and / or the mecC-MRSA gene are provided. mecA / mecC-MRSA nucleic acids other than those exemplified herein can also be used to detect mecA / mecC-MRSA in a sample. For example, functional variants can be evaluated for specificity and / or sensitivity by those skilled in the art using routine methods. Representative functional variants can include, for example, one or more deletions, insertions and / or substitutions in the mecA / mecC-MRSA nucleic acids disclosed herein.

[0034] More specifically, embodiments of the oligonucleotides each comprise a nucleic acid having a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, a substantially identical variant thereof having at least, for example, 80%, 90%, or 95% sequence identity with one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, or a complement of the variant with SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7.

[0035] [Table 1] LCR640 = LightCycler Red 640; IB RQ = Iowa Black RQ

[0036] In one embodiment, to detect mecA / mecC-MRSA in a biological sample suspected of containing mecA / mecC-MRSA, the above sets of mecA-MRSA and mecC-MRSA primers and probes are used. The set of primers and probes can include or consist of primers and probes specific for the mecA-MRSA gene or mecC-MRSA gene nucleic acid sequences, including the nucleic acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7. In another embodiment, the primers and probes for the mecA / mecC-MRSA target can include or consist of functionally active variants of any of the primers and probes of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7.

[0037] Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7 can be identified by using the primers and / or probes in the disclosed methods. Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7 relate to primers and / or probes that provide similar or higher specificity and sensitivity in the described methods or kits as compared to the respective sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7.

[0038] Variants can vary from the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, for example, by one or more nucleotide additions, deletions, or substitutions at the 5' and / or 3' ends of each of the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, such as one or more nucleotide additions, deletions, or substitutions. As detailed above, the primer (and / or probe) may be chemically modified, i.e., the primer and / or probe may contain modified nucleotides or non-nucleotide compounds. Thus, the probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by several modifications, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or phosphate-like moiety, or a combination thereof. For example, a "label" can be attached to the base portion of a "nucleotide" to obtain a "modified nucleotide". The natural base in a "nucleotide" may be replaced, for example, with 7-deazapurine, thereby also obtaining a "modified nucleotide". The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analog") differs from a natural nucleoside by some modification, as outlined above for "modified nucleotides" (or "nucleotide analogs").

[0039] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules encoding mecA-MRSA or mecC-MRSA gene nucleic acid sequences can be designed using a computer program such as OLIGO (Molecular Biology Insights Inc., Cascade, Colorado). Important features in designing oligonucleotides for use as amplification primers include an appropriately sized amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for members of a pair of primers, and the length of each primer (i.e., the primer needs to be long enough to anneal with sequence specificity and initiate synthesis, but not so long that fidelity decreases during oligonucleotide synthesis), but are not limited to these. Typically, oligonucleotide primers are 8 to 50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).

[0040] In addition to a set of primers, the method can use one or more probes to detect the presence or absence of mecA / mecC-MRSA. The term "probe" refers to a synthetic or biologically produced nucleic acid (DNA or RNA) that, by design or selection, contains a specific nucleotide sequence that enables it to specifically (i.e., preferentially) hybridize to mecA-MRSA (target) nucleic acid and / or mecC-MRSA (target) nucleic acid under defined predetermined stringencies. A "probe" can be referred to as a "detection probe" meaning it detects the target nucleic acid.

[0041] In some embodiments, the described mecA-MRSA and mecC-MRSA probes can be labeled with at least one fluorescent label. In one embodiment, the mecA-MRSA and mecC-MRSA probes can be labeled with a donor fluorescent moiety, such as a fluorescent dye, and a corresponding acceptor fluorescent moiety, such as a quencher.

[0042] In one embodiment, the probe comprises or consists of a fluorescent moiety, and the nucleic acid sequence comprises or consists of SEQ ID NO: 3 or 7 (shown without label).

[0043] The design of the oligonucleotide used as a probe can be carried out in a manner similar to that of primer design. Embodiments can use a single probe or a pair of probes for the detection of the amplification product. Depending on the embodiment, the probe(s) used can comprise at least one label and / or at least one quencher moiety. Similar to primers, probes typically have a similar melting temperature, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long as to reduce fidelity during synthesis. Oligonucleotide probes generally have a length of 15 - 30 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides.

[0044] The construct can comprise a vector each containing one of the mecA-MRSA or mecC-MRSA primers and a probe nucleic acid molecule (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7). The construct can be used, for example, as a control template nucleic acid molecule. Vectors suitable for use are commercially available and / or are produced by routine recombinant nucleic acid techniques in the art. The mecA-MRSA and mecC-MRSA nucleic acid molecules can be obtained, for example, by chemical synthesis, direct cloning from mecA / mecC-MRSA, or PCR amplification.

[0045] Constructs suitable for use in the present method typically include, in addition to mecA / mecC-MRSA nucleic acid molecules (e.g., nucleic acid molecules containing one or more of the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7), a sequence encoding a selection marker (e.g., an antibiotic resistance gene) for selecting a desired construct and / or transformant, and an origin of replication. The selection of the vector system usually depends on several factors including, but not limited to, the selection of the host cell, replication efficiency, selectivity, inducibility, and ease of recovery.

[0046] Constructs containing mecA / mecC-MRSA nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is meant to include prokaryotes and eukaryotes, such as yeast, plant, and animal cells. Prokaryotic hosts can include Escherichia coli (E. coli), Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into the host cell using any of the techniques generally known to those of skill in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and virus-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. Additionally, naked DNA can be delivered directly to cells (e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466).

[0047] Polymerase chain reaction (PCR) U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides that can act as starting points for nucleic acid synthesis within the described mecA / mecC-MRSA nucleic acid sequences (e.g., SEQ ID NOs: 1, 2, 4, 5, and 6). Primers can be purified from restriction digests by conventional methods or can be synthetically generated. Although single-stranded primers are preferred for maximum efficiency in amplification, primers can be double-stranded. Double-stranded primers are first denatured. That is, they are treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating. If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturation method, including physical, chemical, or enzymatic means. One method of separating nucleic acid strands involves heating the nucleic acid until it is predominantly denatured (e.g., more than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration, as well as the length and nucleotide composition of the nucleic acid being denatured, but typically range from about 90°C to about 105°C, depending on the characteristics of the reaction, such as temperature and nucleic acid length. Denaturation is typically carried out for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0048] When the double-stranded template nucleic acid is denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence on the described mecA / mecC-MRSA nucleic acid molecule. The annealing temperature is typically from about 35°C to about 65°C (e.g., from about 40°C to about 60°C, from about 45°C to about 50°C). The annealing time can be from about 10 seconds to about 1 minute (e.g., from about 20 seconds to about 50 seconds, from about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient for elongation to occur from the annealed primers to produce a product complementary to the template nucleic acid. The temperature must be sufficient for synthesizing an extension product from each primer annealed to the nucleic acid template, but should not be so high as to denature the extension product from its complementary template (e.g., the temperature for extension generally ranges from about 40°C to about 80°C (e.g., from about 50°C to about 70°C, about 60°C)). The extension time can be from about 10 seconds to about 5 minutes (e.g., from about 30 seconds to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute 30 seconds to about 2 minutes).

[0049] The PCR assay can use mecA / mecC-MRSA nucleic acids such as RNA or DNA (cDNA). The template nucleic acid need not be purified; the template nucleic acid can be a minor fraction of a complex mixture such as the mecA / mecC-MRSA nucleic acid contained in human cells. The mecA / mecC-MRSA nucleic acid molecule can be extracted from biological samples by conventional techniques as described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al., (eds.), 1993, American Society for Microbiology, Washington D.C.). The nucleic acid can be obtained from any number of sources, such as plasmids, or natural sources including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0050] Oligonucleotide primers (e.g., SEQ ID NOs: 1, 2, 4, 5, and 6) are combined with PCR reagents under reaction conditions that induce primer extension. For example, for a strand extension reaction, generally, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5 - 1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO are included. The reaction mixture usually contains dATP, dCTP, dTTP, dGTP, or one or more analogs thereof, each at 150 - 320 μM.

[0051] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of strand separation, annealing, and extension can be repeated as often as necessary to produce the desired amount of amplification product corresponding to the target mecA-MRSA and / or mecC-MRSA nucleic acid molecules. The limiting factors of the reaction are the amounts of primer, heat-stable enzyme, and nucleoside triphosphates present during the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For use in detection, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps are required to amplify sufficient target sequences for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0052] Fluorescence Resonance Energy Transfer (FRET) FRET technology (e.g., U.S. Patent Nos. 4,996,143, 5,565,322, 5,849,489, 6,162,603) is based on the concept that energy transfer occurs between two fluorescent moieties such that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are placed within a certain distance of each other, they can be visualized or otherwise detected and / or quantified. Typically, when the donor is excited by light radiation of a suitable wavelength, it transfers energy to the acceptor. Typically, the acceptor re - emits the transferred energy in the form of light radiation of a different wavelength. In certain systems, non - fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule that includes a substantially non - fluorescent donor moiety (see, e.g., U.S. Patent No. 7,741,467).

[0053] In one example, an oligonucleotide probe can contain a donor fluorescent moiety and a corresponding quencher that may or may not be fluorescent and dissipates energy transmitted in a form other than light. When the probe is intact, energy transfer typically occurs between the two fluorescent moieties such that fluorescence emission from the donor fluorescent moiety is quenched. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase, such that the fluorescence emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ) (Biosearch Technologies, Inc., Novato, CA), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, IA), and BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc, Dexter, MI).

[0054] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at specific positions determined by the complementarity of the oligonucleotide probe to the mecA / mecC-MRSA target nucleic acid sequence. When the oligonucleotide probe hybridizes to the amplification product nucleic acid at the appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0055] Fluorescence analysis can be performed using, for example, a photon-counting epi-fluorescence microscope system (equipped with a suitable dichroic mirror and a filter for monitoring fluorescence emission in a specific range), a photon-counting photomultiplier tube system, or a fluorometer. Excitation to initiate energy transfer or to enable direct detection of the phosphor can be carried out using an argon ion laser, a high-intensity mercury (Hg) arc lamp, an optical fiber light source, or other high-intensity light sources appropriately filtered for the desired range of excitation.

[0056] As used herein with respect to "corresponding" in the donor fluorescent moiety and the corresponding acceptor fluorescent moiety, it refers to an acceptor fluorescent moiety having an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Thus, efficient non-radiative energy transfer can be generated therebetween.

[0057] The fluorescent donor moiety and the corresponding acceptor moiety are generally selected for (a) high-efficiency Forster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift of the emission to the red portion (>600 nm) of the visible spectrum as much as possible; and (d) a shift of the emission to a higher wavelength than the Raman water fluorescence emission caused by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety having its maximum excitation wavelength near the laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high absorption coefficient, a high quantum yield, and good overlap with the excitation spectrum of the acceptor fluorescent moiety corresponding to its fluorescence emission can be selected. A corresponding acceptor fluorescent moiety having a high absorption coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion (>600 nm) of the visible spectrum can be selected.

[0058] In FRET technology, typical donor fluorescent moieties that can be used with various acceptor fluorescent moieties include fluorescein, lucifer yellow, B-phycoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Typical acceptor fluorescent moieties include LC Red640, LC Red705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosin isothiocyanate, fluorescein, diethylenetriaminepentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. The donor fluorescent moiety and the acceptor fluorescent moiety can be obtained, for example, from Molecular Probes (Eugene, Oregon) or Sigma Chemical Co. (St. Louis, Missouri).

[0059] The donor fluorescent moiety and the acceptor fluorescent moiety can be attached to an appropriate probe oligonucleotide via a linker arm. Since the linker arm affects the distance between the donor fluorescent moiety and the acceptor fluorescent moiety, the length of each linker arm is important. The length of the linker arm can be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Generally, the linker arm is about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching the linker arm to a specific nucleotide base and for attaching the fluorescent moiety to the linker arm.

[0060] An acceptor fluorescent moiety such as LC Red640 can be combined with an oligonucleotide containing an amino linker (e.g., a C6 - aminophosphoramidite available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to generate, for example, an LC Red640 - labeled oligonucleotide. Linkers commonly used to attach a donor fluorescent moiety such as fluorescein to an oligonucleotide include a thiourea linker (derived from FITC, e.g., fluorescein - CPG manufactured by Glen Research or ChemGene (Ashland, Mass.)), an amide - linker (derived from fluorescein - NHS - ester, such as CX - fluorescein - CPG manufactured by BioGenex (San Ramon, Calif.)), or 3’ - amino - CPG which requires the binding of fluorescein - NHS - ester after oligonucleotide synthesis.

[0061] Detection of mecA / mecC - MRSA The present disclosure provides a method for detecting the presence or absence of mecA / mecC - MRSA in a biological or non - biological sample. The method provided circumvents problems of sample contamination, false negatives, and false positives. The method includes at least one cycling step of amplifying a portion of a mecA - MRSA and / or mecC - MRSA target nucleic acid molecule from the sample using a plurality of pairs of mecA - MRSA and / or mecC - MRSA primers, and performing a FRET detection step. The plurality of cycling steps are preferably performed in a thermocycler. To detect the presence of mecA / mecC - MRSA, the method can be carried out using mecA - MRSA and mecC - MRSA primers and probes, and the detection of mecA / mecC - MRSA indicates the presence of mecA - MRSA and / or mecC - MRSA in the sample.

[0062] As described herein, amplification products can be detected using labeled hybridization probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of amplification products and thus the presence or absence of mecA / mecC-MRSA. TaqMan® technology utilizes, for example, a single-stranded hybridization probe labeled with one fluorescent dye and one quencher, which may or may not be fluorescent. When the first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is generally a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is then degraded, for example, by the 5' to 3' nuclease activity of Taq polymerase during the subsequent extension step. As a result, the fluorescent moiety and the quencher moiety become spatially separated from each other. Consequently, when the first fluorescent moiety is excited in the absence of the quencher, fluorescence emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for performing the methods described herein for detecting the presence or absence of mecA / mecC-MRSA in a sample.

[0063] The presence of amplification products can also be detected using real-time PCR with molecular beacons combined with FRET. Molecular beacon technology uses hybridization probes labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and the fluorescent labels are typically placed at each end of the probe. Molecular beacon technology uses probe oligonucleotides having sequences that allow for secondary structure formation (e.g., hairpins). As a result of secondary structure formation within the probe, when the probe is in solution, both fluorescent moieties are spatially proximal. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted and the fluorescent moieties are separated from each other, thereby allowing the emission of the first fluorescent moiety to be detected after excitation with light of an appropriate wavelength.

[0064] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to each other within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, such as fluorescein, is excited at 470 nm by the light source of a LightCycler® instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, such as LightCycler®-Red640 (LC Red640) or LightCycler®-Red705 (LC Red705). The acceptor fluorescent moiety then emits light at a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can occur only when the fluorescent moieties are directly locally proximal and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of mecA-MRSA or mecC-MRSA genomes). When amplification of the mecA / mecC-MRSA target nucleic acid occurs and amplification products are produced, the hybridization step results in a detectable signal based on FRET between the members of the probe pair.

[0065] Generally, the presence of FRET indicates the presence of mecA / mecC-MRSA in the sample, and the absence of FRET indicates the absence of mecA / mecC-MRSA in the sample. However, insufficient specimen collection, transport delays, inappropriate transport conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results. Using the methods disclosed herein, for example, detection of FRET within 45 cycling steps indicates infection with mecA-MRSA or mecC-MRSA.

[0066] Representative biological samples that can be used in the practice of this method include, but are not limited to, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods for collecting and preserving biological samples are known to those of ordinary skill in the art. By processing biological samples (e.g., by nucleic acid extraction methods and / or kits known in the art), mecA / mecC-MRSA nucleic acids can be released, or in some cases, biological samples can be brought into direct contact with PCR reaction components and appropriate oligonucleotides.

[0067] Melting curve analysis is an additional step that can be included in the cycle profile.

[0068] Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which half of the DNA double strands separate into single strands. The melting temperature of DNA mainly depends on its nucleotide composition. Therefore, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, the annealing temperature of the probe can be determined by detecting the temperature at which the signal is generated. The presence or absence of mecA / mecC-MRSA in the sample can be confirmed by the melting temperatures of the mecA-MRSA and mecC-MRSA probes from the mecA / mecC-MRSA amplification product.

[0069] Between each thermocycler run, control samples can also be cycled in the same way. Positive control samples can amplify a target nucleic acid control template (other than the amplification product of the described target gene), for example, using control primers and control probes. Positive control samples can also amplify, for example, a plasmid construct containing the target nucleic acid molecule. Such plasmid controls can be amplified in a separate sample run internally (e.g., within the sample) or alongside the patient's sample using the same primers and probes as those used for the detection of the intended target. Such controls are indicators of the success or failure of the amplification, hybridization, and / or FRET reaction. Each thermocycler run can also include, for example, a negative control lacking the target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false positive signals. Therefore, control reactions can easily determine, for example, the ability of the primers to anneal sequence-specifically and initiate elongation, as well as the ability of the probes to hybridize sequence-specifically and generate FRET.

[0070] In one embodiment, the method includes a step of avoiding contamination. For example, enzymatic methods using uracil-DNA glycosylase are described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next thermocycler run.

[0071] This method can be implemented using a conventional PCR method combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications: International Publication Nos. 97 / 46707, 97 / 46714, and 97 / 46712 disclose real-time PCR used in LightCycler® technology.

[0072] LightCycler® can be operated using a PC workstation and can utilize the Windows NT operating system. Signals from samples are obtained when the machine sequentially positions capillaries on the optical unit. The software can display fluorescence signals in real time immediately after each measurement. The fluorescence acquisition time is 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus the number of cycles can be continuously updated for all samples. The generated data can be saved for further analysis.

[0073] As an alternative to FRET, amplification products can be detected using a double-stranded DNA-binding dye such as a fluorescent DNA-binding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). When interacting with double-stranded nucleic acids, such fluorescent DNA-binding dyes emit a fluorescence signal after excitation with light of an appropriate wavelength. Also, double-stranded DNA-binding dyes such as nucleic acid intercalating dyes can be used. When using a double-stranded DNA-binding dye, melting curve analysis is usually performed to confirm the presence of amplification products.

[0074] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0075] Manufactured article / Kit Embodiments of the present disclosure further provide a manufactured article or kit for detecting mecA / mecC-MRSA. The manufactured article may include primers and probes used for detecting mecA / mecC-MRSA, together with a suitable packaging material. Representative primers and probes for the detection of mecA / mecC-MRSA may hybridize to a mecA / mecC-MRSA target nucleic acid molecule. Further, the kit may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as a solid support, buffer, enzyme, and DNA standard. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to a mecA / mecC-MRSA target nucleic acid molecule are provided.

[0076] The manufactured article may also include one or more fluorescent moieties for labeling the probe, or may label the probe supplied with the kit. For example, the manufactured article may include donor and / or acceptor fluorescent moieties for labeling mecA-MRSA and mecC-MRSA probes. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0077] The manufactured article may also include an attached document or attached document label having instructions for using the mecA-MRSA and mecC-MRSA primers and probes for detecting mecA / mecC-MRSA in a sample. The manufactured article may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents for preventing contamination) for performing the methods disclosed herein. Such reagents may be specific to one of the commercially available devices described herein.

[0078] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the invention described in the claims.

Example

[0079] The following examples and drawings are provided to assist in the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that changes can be made to the defined procedures without departing from the spirit of the invention.

[0080] Example 1 MecA / mecC-MRSA gene target Figure 1 shows the relative positions of some target genes used for the detection of methicillin-resistant Staphylococcus aureus (MRSA). The primers and probes of the present disclosure target the mecA / mecC gene.

[0081] Example 2 PCR experimental conditions Real-time PCR detection of the mecA-MRSA target gene or mecC-MRSA target gene was performed using either the cobas® 4800 system or the cobas® 6800 / 8800 system platform (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of the amplification reagents are shown below:

[0082]

Table 2

[0083] The following table shows a typical thermal profile used for the PCR amplification reaction:

[0084]

Table 3

[0085] Example 3 Experimental results Figure 2 shows the PCR growth curves of experiments using mecA primers and probes from a standard oligonucleotide vendor against MRSA strain 12770 known to contain the mecA gene. Despite the detection of the mecA gene, the growth curve was not S-shaped, and as a result, the Ct value (threshold cycle) could not be determined, and quantification of the target gene concentration could not be performed. In contrast, in the PCR experiment using the mecA forward primer of SEQ ID NO: 1, the mecA reverse primer of SEQ ID NO: 2, and the mecA probe of SEQ ID NO: 3, detection of amplification products was observed only in MRSA strains (strains 10714, 10817, and 12270) containing the mecA gene, and not in the MRSA strain (strain 12756) containing the mecC gene or the methicillin-susceptible Staphylococcus aureus (MSSA) strain 10853. Figure 3 shows the growth curves of this PCR experiment, and the determined Ct values are shown in Table IV.

[0086] [Table 4] In another PCR experiment, mecC-containing MRSA strains 12756 and 12765 were amplified and detected using the mecC forward primer of SEQ ID NO: 4 or the mecC forward primer of SEQ ID NO: 5 and the mecC reverse primer of SEQ ID NO: 6, and detected using the mecC probe of SEQ ID NO: 7. The results are shown in Table V and Figure 4.

[0087]

Table 5

[0088]

Table 6

Claims

1. 1. A method for detecting mecA-containing methicillin-resistant Staphylococcus aureus (mecA-MRSA) in a sample, comprising: - if mecA-MRSA is present in the sample, performing an amplification step comprising contacting the sample with a forward primer comprising the nucleotide sequence of SEQ ID NO: 1 and a reverse primer comprising the nucleotide sequence of SEQ ID NO: 2 to produce an amplification product; - performing a hybridization step comprising contacting said amplification product with one or more detectable mecA-MRSA probes, wherein one of said one or more detectable mecA-MRSA probes comprises the sequence of SEQ ID NO: 3 or its complement; and - detecting the presence or absence of the amplified amplification product, wherein the presence of the amplified amplification product indicates the presence of mecA-MRSA in the sample and the absence of the amplified amplification product indicates the absence of mecA-MRSA in the sample. A method comprising:

2. - the hybridizing step comprises contacting the amplification product with one of the one or more mecA-MRSA probes labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; 2. The method of claim 1, wherein the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety of the probe, wherein the presence or absence of fluorescence FRET indicates the presence or absence of mecA-MRSA in the sample.

3. 3. The method of claim 2, wherein the donor fluorescent moiety and the corresponding acceptor fluorescent moiety are within 8 nucleotides of each other on the probe.

4. The method of claim 2 or 3, wherein the acceptor fluorescent moiety is a quencher.

5. The method according to any one of claims 2 to 4, wherein the amplification step uses a polymerase enzyme having 5' to 3' nuclease activity.

6. - if mecC-MRSA is present in the sample, further contacting the sample in the amplification step with a forward primer comprising the nucleotide sequence of SEQ ID NO: 4 or 5 and a reverse primer comprising the nucleotide sequence of SEQ ID NO: 6 to produce an amplification product; - further contacting the amplification product of the hybridizing step with one or more detectable mecC-MRSA probes, wherein one of the one or more detectable mecC-MRSA probes comprises the sequence of SEQ ID NO: 7 or its complement; and - detecting the presence or absence of said amplified amplification product, wherein the presence of said amplified amplification product indicates the presence of mecC-MRSA in said sample and the absence of said amplified amplification product indicates the absence of mecC-MRSA in said sample.

6. The method of claim 2, further comprising detecting mecC-containing methicillin-resistant Staphylococcus aureus (mecC-MRSA) in the sample in the same reaction by:

7. 7. The method of claim 6, wherein the mecC-MRSA probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety that are different from the donor fluorescent moiety and the corresponding acceptor fluorescent moiety of the labeled mecA-MRSA probe.

8. A kit for detecting nucleic acids of mecA-containing Staphylococcus aureus (mecA-MRSA), comprising: a first oligonucleotide comprising the sequence SEQ ID NO: 1; a second oligonucleotide comprising the sequence SEQ ID NO: 2; and a third detectably labeled oligonucleotide comprising SEQ ID NO: 3 or its complement, configured to hybridize to the amplicon generated by said first oligonucleotide and said second oligonucleotide; Includes a kit.

9. 9. The kit of claim 8, wherein the third detectably labeled oligonucleotide comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.

10. The kit of claim 9 , wherein the acceptor fluorescent moiety is a quencher.

11. 11. The kit of claim 9 or 10, further comprising nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase.

12. 12. The kit according to any one of claims 9 to 11, further for detecting nucleic acids of mecC-containing Staphylococcus aureus (mecC-MRSA), comprising: a fourth oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 4 and 5; a fifth oligonucleotide comprising the sequence SEQ ID NO: 6; and a sixth detectably labeled oligonucleotide comprising SEQ ID NO: 7 or its complement, configured to hybridize to the amplicon generated by the fourth oligonucleotide and the fifth oligonucleotide; The kit further comprises:

13. The kit described in claim 12, wherein the sixth detectably labeled oligonucleotide is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety that are different from the donor fluorescent moiety and the corresponding acceptor fluorescent moiety of the third detectably labeled oligonucleotide.

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