Compositions and methods for detecting vanA and / or vanB genes associated with multidrug resistance - Patents.com

Abstract

A method is described for the rapid detection of the presence or absence of bacteria with vanA and / or vanB resistance mechanisms in a biological or non-biological sample. The method can include carrying out an amplification step, a hybridization step, and a detection step. Additionally, primers, probes targeting the vanA and vanB genes are provided along with kits designed for the detection of vanA and vanB.

Description

[Technical field]

[0001] The present disclosure relates to the field of bacterial diagnostics, and more particularly to the detection of vancomycin-resistant bacteria, such as Staphylococcus aureus, that contain vanA and / or vanB nucleic acid sequences. [Background technology]

[0002] Vancomycin is a glycopeptide antibiotic with strong activity against many Gram-positive organisms. In many treatment regimens, vancomycin is considered a "drug of last resort" (DoLR), meaning that it is used only after all other drug options have failed. Because vancomycin is a DoLR, it is important to identify whether a patient is colonized or infected with bacteria that are resistant to vancomycin.

[0003] The van genes are a resistance mechanism that confers resistance to vancomycin by altering peptidoglycan synthesis, and their presence has been documented for many Gram-positive species. Multiple variants / types of the genes (vanA / B / C / D / E / G) exist, each of which holds unique characteristics in terms of resistance profile, transmissibility, and prevalence. In particular, the vanA and vanB genes are of interest because they can reside on transposons (i.e., resistance genes can be mobile) and because they are prevalent worldwide. Clinically relevant examples of vancomycin-resistant bacteria include vancomycin-resistant Enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA).

[0004] The vanA gene encodes a protein that confers high-level resistance to vancomycin and teicoplanin (Arthur et al., 1993, 1996). Expression of the vanA gene is inducible by vancomycin or teicoplanin. VanA-type glycopeptide resistance has been described in several Enterococcus species (for review, see Mendez-Alvarez et al., 2000). The vanB gene confers resistance to various concentrations of vancomycin but not teicoplanin (Baptista et al., 1996; Evers & Courvalin, 1996) and is inducible only by vancomycin but not by teicoplanin. VanB-type glycopeptide resistance has been described in Enterococcus faecalis and Enterococcus faecium. Recently, the genomes of some methicillin-resistant Staphylococcus aureus (MRSA) strains have evolved to generate vancomycin-resistant Staphylococcus aureus (VRSA) strains as a result of acquiring the vanA gene from Enterococci. Thus, there is a need in the art for a rapid and reliable method to specifically detect bacteria containing both vanA and vanB in a specific and sensitive manner. Summary of the Invention

[0005] Some aspects of the present invention relate to a method for rapid detection of the presence or absence of bacteria with vanA and / or vanB resistance mechanisms in biological or non-biological samples, for example, multiplex detection of vanA and vanB by real-time polymerase chain reaction in a single test tube. The embodiments include a method for detection of vanA and vanB resistance mechanisms by determining the presence of vanA and / or vanB genes, comprising performing at least one cycling step, which may include an amplifying step and a hybridizing step. Furthermore, the embodiments include primers, probes, and kits designed for detection of vanA and vanB resistance mechanisms in a single tube. The detection method is designed to target vanA and vanB genes, which allows detection of vancomycin-resistant strains in a single test. In one embodiment, the vancomycin-resistant strain is Staphylococcus aureus.

[0006] In one aspect, a method is provided for detecting bacteria having vanA and / or vanB genes in a sample, comprising: performing an amplification step comprising contacting the sample with a vanA forward and reverse primer set and a vanB forward and reverse primer set to generate an amplification product if vanA and / or vanB genes are present in the sample; performing a hybridization step comprising contacting the amplification product with one or more detectable vanA probes and one or more detectable vanB probes; and detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of the vanA and / or vanB genes in the sample and the absence of the amplification product indicates the absence of the vanA and / or vanB genes in the sample; wherein the vanA primer set comprises an oligonucleotide sequence of SEQ ID NO: 1 or 2, or a complement thereof. the set of vanB primers comprises or consists of a forward primer comprising or consisting of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 7, 11 and 12 or its complement, and a reverse primer comprising or consisting of an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 8, 9, 13 and 14 or its complement; the one or more detectable vanA probes comprises or consists of the oligonucleotide sequence of SEQ ID NO: 5 or its complement, and the one or more detectable vanB probes comprises or consists of the oligonucleotide sequence of SEQ ID NO: 10 or 15 or its complement.In some embodiments, the hybridizing step includes contacting the amplification product with a detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; the detecting step includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety of the probe, the presence or absence of fluorescent FRET indicating the presence or absence in the sample. In some embodiments, the amplifying step uses a polymerase enzyme with 5' to 3' nuclease activity. In some embodiments, the donor fluorescent moiety and the corresponding acceptor fluorescent moiety are within 8 nucleotides of each other on the probe. In some embodiments, the acceptor fluorescent moiety is a quencher. In one embodiment, the vanA primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:2, and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:4; the vanB primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:7, and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:9; one or more detectable vanA probes comprise the oligonucleotide sequence of SEQ ID NO:5, and one or more detectable vanB probes comprise the oligonucleotide sequence of SEQ ID NO:10.

[0007] In another aspect, the present invention relates to a set of primers and probes for the amplification and detection of vanA gene target sequences, comprising or consisting of (at least) one forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 1 or 2 or its complement; (at least) one reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 3 or 4 or its complement; and (at least) one detectable probe for the detection of vanA amplification products comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 5 or its complement. In one embodiment, the detectable probe comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety. In some embodiments, the acceptor fluorescent moiety is a quencher.

[0008] In another aspect, the present invention relates to a primer and probe set for the amplification and detection of vanB gene target sequences, comprising or consisting of (at least) one forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 6, 7, 11 and 12 or its complement, (at least) one reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 8, 9, 13 and 14 or its complement, and (at least) one detectable probe for the detection of vanB amplification products comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 10 and 15 or its complement. In one embodiment, the detectable probe comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety. In some embodiments, the acceptor fluorescent moiety is a quencher.

[0009] In another aspect, the present invention relates to a set of primers for the amplification of vanA gene targets, comprising at least one primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 1, 2, 3, and 4, or its complement, and a detectable probe for the detection of vanA amplification products comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 5, or its complement; and / or a set of primers for the amplification of vanB gene targets, comprising at least one primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 6, 7, 8, 9, 11, 12, 13, and 14, or its complement, and a detectable probe for the detection of vanB amplification products comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 10 and 15, or its complement. In one embodiment, the detectable probe comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety. In some embodiments, the acceptor fluorescent moiety is a quencher.

[0010] In yet another aspect, the present disclosure provides an oligonucleotide comprising or consisting of a sequence of nucleotides selected from SEQ ID NOs: 1-15 or its complement, the oligonucleotide comprising 100 or less nucleotides. In another embodiment, 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%, etc.) to one of SEQ ID NOs: 1-15 or its complement, the oligonucleotide comprising 100 or less nucleotides. Generally, the oligonucleotides may be primer nucleic acids, probe nucleic acids, etc. in these embodiments. In some of these embodiments, the oligonucleotide comprises 40 or less nucleotides (e.g., 35 or less nucleotides, 30 or less nucleotides, etc.). In some embodiments, the oligonucleotide comprises at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to unmodified nucleotides. 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 mutation. "Conservatively modified variations" or simply "conservative variations" of a particular nucleic acid sequence refer to nucleic acids that code for identical or essentially identical amino acid sequences, or essentially identical sequences if the nucleic acid does not code for an amino acid sequence. Those skilled in the art will recognize that individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2%, or 1%) in an encoded sequence are "conservatively modified variations" where the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid. In some embodiments, such oligonucleotides are suitable for use in the methods, primer and probe sets, and kits according to the invention.

[0011] In one embodiment, the amplification can use a polymerase enzyme with 5' to 3' nuclease activity. Thus, the first and second fluorescent moieties can be within 8 nucleotides of each other along the length of the probe. In another embodiment, the vanA and vanB probes contain nucleic acid sequences that allow for the formation of secondary structures. The formation of such secondary structures generally results in spatial proximity between the first and second fluorescent moieties. According to this method, the second fluorescent moiety on the probe can be a quencher.

[0012] In a further aspect, the present invention provides a kit for detecting one or more nucleic acids of vanA and / or vanB resistance mechanisms. The kit may include multiple sets of vanA and / or vanB primers specific for amplifying vanA and / or vanB gene targets; and one or more detectable vanA and / or vanB probes specific for detecting vanA and / or vanB amplification products.

[0013] In one aspect, a kit for detecting nucleic acid of vanA-containing Staphylococcus aureus is provided, comprising a primer-probe set comprising or consisting of (at least) one forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 1 or 2 or its complement, (at least) one reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 3 or 4 or its complement, and (at least) one detectable probe for detection of vanA amplification products comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 5 or its complement, and further comprising at least one of nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase.

[0014] In one aspect, a kit for detecting nucleic acid of vanB-containing Staphylococcus aureus is provided, comprising a primer-probe set comprising or consisting of (at least) one forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 6, 7, 11, and 12, or its complement, (at least) one reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 8, 9, 13, and 14, or its complement, and (at least) one detectable probe for detection of vanB amplification products comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 10 and 15, or its complement, and further comprising at least one of nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase.

[0015] In another aspect, a kit for detecting vanA-containing and / or vanB-containing S. aureus nucleic acid is provided, the kit comprising a primer and probe set for amplifying and detecting vanA nucleic acid, and a primer and probe set for amplifying and detecting vanB nucleic acid, further comprising at least one of nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase. As used herein, the vanA primer and probe set may comprise or consist of (at least) one forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 1 or 2 or its complement, (at least) one reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 3 or 4 or its complement, and (at least) one detectable probe for detection of vanA amplification product comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 5 or its complement, and / or vanB. The primer and probe set may comprise or consist of (at least) one forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 6, 7, 11 and 12 or its complement, (at least) one reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 8, 9, 13 and 14 or its complement, and (at least) one detectable probe for detection of vanB amplification products comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 10 and 15 or its complement.

[0016] In some embodiments, the kits may include probes already labeled with donor and corresponding acceptor fluorescent moieties, or may include fluorescent moieties for labeling the probes. The kits may also include nucleoside triphosphates, nucleic acid polymerase, and buffers required for the function of the nucleic acid polymerase, packaged in a single tube or combined in one tube. The kits may also include inserts and instructions for using the primers, probes, and fluorescent moieties to detect the presence or absence of vanA and / or vanB genes in a sample.

[0017] Another aspect of the invention relates to a method for the rapid detection of Staphylococcus aureus (S. aureus) with vancomycin resistance (VRSA), methicillin resistance (MRSA), or both VRSA and MRSA. The method comprises contacting a sample with: a vanA forward and reverse primer set and a vanB forward and reverse primer set to generate an amplification product if vanA and / or vanB genes are present in the sample; a forward and reverse primer set to generate an amplification product of S. aureus capsular polysaccharide enzyme (CPE) if S. aureus is present in the sample (as disclosed in U.S. Pat. No. 9,034,581, which is incorporated by reference in its entirety); a forward and reverse primer set to generate an amplification product of the right end junction of the Staphylococcus aureus chromosomal cassette mec (SCCmec) mobile genetic element if SCCmec is present in the sample; and a mecA forward and reverse primer set to generate an amplification product of the right end junction of the Staphylococcus aureus chromosomal cassette mec (SCCmec) mobile genetic element if mecA and / or mecC genes are present in the sample. performing an amplification step comprising contacting a set of vanA and vanB probes and a set of mecC forward and reverse primers to generate an amplification product; performing a hybridization step comprising contacting the amplification product with one or more detectable vanA probes, one or more detectable vanB probes, one or more detectable CPE probes, one or more detectable SCCmec probes, one or more detectable mecA probes, and one or more detectable mecC probes; and detecting the presence or absence of an amplification product, wherein the presence of an amplification product of the vanA genes and / or vanB genes and the CPE genes indicates the presence of VRSA in the sample, and the presence of an amplification product of the SCCmec elements, the mecA genes and / or the mecC genes and the CPE genes indicates the presence of MRSA in the sample.

[0018] In one embodiment, the set of vanA forward and reverse primers for the amplification of the vanA gene comprises at least one primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 1, 2, 3, and 4, or its complement, and one or more detectable probes for the detection of the vanA amplification product comprise or consist of an oligonucleotide sequence of SEQ ID NO: 5, or its complement. In one embodiment, the set of vanB forward and reverse primers for the amplification of the vanB gene comprises at least one primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 6, 7, 8, 9, 11, 12, 13, and 14, or its complement, and one or more detectable probes for the detection of the vanB amplification product comprise or consist of an oligonucleotide sequence selected from SEQ ID NOs: 10 and 15, or its complement, or its complement. In one embodiment, the vanA primer and probe set may comprise or consist of (at least) one forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 1 or 2 or its complement, (at least) one reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 3 or 4 or its complement, and (at least) one detectable probe for the detection of vanA amplification products comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 5 or its complement, and / or The set of B primers and probes may comprise or consist of (at least) one forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 6, 7, 11 and 12 or its complement, (at least) one reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 8, 9, 13 and 14 or its complement, and (at least) one detectable probe for detection of vanB amplification products comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 10 and 15 or its complement.In certain embodiments, the vanA primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:2 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:4; the vanB primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:7 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:9; the one or more detectable vanA probes comprise the oligonucleotide sequence of SEQ ID NO:5 and the one or more detectable vanB probes comprise the oligonucleotide sequence of SEQ ID NO:10. In some embodiments, the S. aureus CPE forward and reverse primer set for amplification of the S. aureus CPE gene comprises at least one primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs:16 and 17 or a complement thereof, and the one or more detectable probes for detection of the S. aureus CPE amplification product comprise or comprise the oligonucleotide sequence of SEQ ID NO:18 or a complement thereof. In some embodiments, the CPE primer set comprises or consists of a forward primer that comprises or consists of the oligonucleotide sequence of SEQ ID NO: 16 or its complement and a reverse primer that comprises or consists of the oligonucleotide sequence of SEQ ID NO: 17 or its complement, and the one or more detectable CPE probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 18 or its complement. In one embodiment, the SCCmec forward and reverse primer set for amplification of SCCmec elements comprises at least one primer that comprises or consists of an oligonucleotide sequence selected from SEQ ID NOs: 19, 20, 21, 22, 23 and 24 or its complement, and the one or more detectable probes for detection of SCCmec amplification products comprise or consist of the oligonucleotide sequence of SEQ ID NO: 25 or its complement.In some embodiments, the SCCmec primer set comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 19 and 20 or a complement thereof and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 21, 22, 23 and 24 or a complement thereof, and the one or more detectable vanB probes comprise the oligonucleotide sequence of SEQ ID NO: 25 or a complement thereof. In one embodiment, the mecA forward and reverse primer set for amplification of the mecA gene comprises at least one primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 26 and 27 or a complement thereof, and the one or more detectable probes for detection of the mecA amplification product comprise or consist of the oligonucleotide sequence of SEQ ID NO: 28 or a complement thereof. In some embodiments, the mecA primer set comprises a forward primer comprising an oligonucleotide sequence of SEQ ID NO: 26 or a complement thereof and a reverse primer comprising an oligonucleotide sequence of SEQ ID NO: 27 or a complement thereof, and the one or more detectable mecA probes comprise the oligonucleotide sequence of SEQ ID NO: 28 or a complement thereof. In one embodiment, the mecA forward and reverse primer set for amplification of the mecC gene consists of at least one primer that comprises or consists of an oligonucleotide sequence selected from SEQ ID NOs: 29 and 30, or a complement thereof, and the one or more detectable probes for detection of the mecC amplification product comprise or consist of the oligonucleotide sequence of SEQ ID NO: 31, or a complement thereof. In some embodiments, the mecC primer set comprises a forward primer that comprises the oligonucleotide sequence of SEQ ID NO: 29, or a complement thereof, and a reverse primer that comprises the oligonucleotide sequence of SEQ ID NO: 30, or a complement thereof, and the one or more detectable mecC probes comprise the oligonucleotide sequence of SEQ ID NO: 31, or a complement thereof.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present subject matter, and suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and are 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.

[0020] The 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 become apparent from the drawings, the detailed description, and the claims. [Brief description of the drawings]

[0021] [Figure 1] 1 shows the results of a PCR assay using non-benzylated vanA and vanB primers as described in Example 3. [Diagram 2] 1 shows the results of a PCR assay using benzylated vanA and vanB primers as described in Example 3. [Diagram 3] 1 shows the results of a multiplex PCR assay using primer and probe sets targeting vanA, vanB, CPE, SCCmec OrfX, mecA and mecC in various S. aureus and Enterococcus strains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Diagnosis of bacteria with vanA and / or vanB resistance mechanisms by nucleic acid amplification provides a method for rapid and accurate detection of bacterial infection. Described herein are real-time assays for detecting vanA and vanB genes in a sample. Primers and probes for detecting vanA and / or vanB are provided, as are articles of manufacture or kits containing such primers and probes. The increased sensitivity of real-time PCR for detection of vanA and / or vanB compared to other methods, and improved features of real-time PCR, including sample containment and real-time detection of amplification products, allow for the implementation of this technology for routine diagnosis of vanA and / or vanB infections, including but not limited to vancomycin-resistant Enterococcus (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA), in clinical laboratories.

[0023] Glycopeptide resistance in enterococci is phenotypically and genotypically heterogeneous. The genes responsible for high-level inducible resistance to vancomycin and teicoplanin (VanA phenotype) are carried by the 10,851-bp Tn1546 transposon. Subsequent transfer by transposition and conjugation of Tn1546 to self-mobilizable plasmids is likely responsible for the spread of this type of resistance. Nine polypeptides are encoded by Tn1546 belonging to five functional groups: transposition functions (ORF1 and ORF2), control of resistance gene expression (VanR and VanS), synthesis of the depsipeptide d-Ala-d-lactate (VanH and VanA), hydrolysis of d-Ala-d-Ala-containing peptidoglycan precursors (VanX and VanY), and low-level teicoplanin resistance (VanZ). VanB type resistance (variable levels of resistance to vancomycin and sensitivity to teicoplanin) is also due to the production of d-Ala-d-Lac. The VanB ligase of VanB type strains is structurally and functionally similar to VanA. The vanB gene was found on the composite transposon Tn1547, which was part of a larger conjugative chromosomally located element (90 to 250 kb). In contrast to acquired VanA and VanB type resistance, VanC type resistance (low levels of resistance to vancomycin and sensitivity to teicoplanin) is an intrinsic property of motile enterococci. Resistance in these species is due to the synthesis of the dipeptide d-Ala-d-Ser by VanC ligase, which leads to the production of a cell wall precursor with reduced affinity for vancomycin.

[0024] Both the methicillin resistance gene mecA and its homolog mecC encode penicillin-binding proteins with reduced affinity for the β-lactam ring (the main active site of β-lactam antibiotics such as penicillins, cephalosporins and carbapenems), an altered methicillin-resistant penicillin-binding protein (PBP2a or PBP2') that is absent in susceptible strains and is likely acquired from distantly related species (Guignard et al., 2005, Curr Opin Pharmacol 5(5):479-89). Both mecA and mecC are carried on a mobile genetic element, the Staphylococcal chromosomal cassette mec (SCCmec) in MRSA strains. SCC elements also occur in susceptible S. aureus, but they do not carry mecA or mecC genes, nor do they carry non-functional mecA or mecC genes. Such strains may have the same right extremity junction and therefore may be a source of false positive results.

[0025] MRSA detection from nasal specimens by detecting mecA or mecC genes and S. aureus specific genes will sometimes lead to low positive predictive value (PPV) due to the presence of variable amounts of both non-resistant S. aureus and methicillin-resistant coagulase negative Staphylococci (MRCoNS). Due to the presence of both targets, their combination cannot be distinguished from MRSA. Depending on the prevalence of MRSA, this situation leads to up to 30% false positive results. For better PPV, the selected target needs to be unique for MRSA. The only target currently known is the Staphylococcus chromosomal cassette amplifying transposon integration (SCCmec).

[0026] Detection of mecA / mecC-containing S. aureus (mecC-MRSA) utilizes a strategy that generates an amplicon at the RE junction between the S. aureus orfX gene and the SCCmec carrying the mecA or mecC genes that confer resistance to methicillin. To achieve this, one primer is anchored in a highly conserved region of the S. aureus orfX gene (orfX primer) and a second primer is located within the non-conserved RE junction of SCCmec (RE primer). The amplicon resulting from the two primers spans a portion of the orfX gene and a portion of the SCCmec. Due to the non-homologous nature of SCCmec at the RE junction, multiple different RE primers are required to achieve maximum coverage of unique MRSA strains. This type of identification and detection of mecA / mecC-MRSA has been described by several groups, for example, in 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., and 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 previously mentioned, this strategy can be a source of false positive results if the mecA or mecC genes are deleted (in whole or in part) or non-functional.

[0027] The term "amplifying" as used herein refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., vanA and / or vanB). 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 cofactors for optimal activity of the polymerase enzyme (e.g., MgCl2 and / or KCl).

[0028] The term "primer" as used herein is known to those skilled in the art and refers to oligomeric compounds, mainly oligonucleotides, but also modified oligonucleotides that can "prime" DNA synthesis by template-dependent DNA polymerases, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group to which a "nucleotide" can be further attached by the template-dependent DNA polymerase that establishes a 3' to 5' phosphodiester bond, thereby using a deoxynucleoside triphosphate and releasing pyrophosphate. Thus, there is no fundamental difference between a "primer", an "oligonucleotide", or a "probe", except perhaps in the intended function.

[0029] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. Hybridization conditions typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0030] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0031] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when exposed to high temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. 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. Nonetheless, non-thermostable polymerases can be used in PCR assays if the enzyme is replenished.

[0032] The term "the complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0033] The term "extension" or "lengthening" when used in reference to a nucleic acid refers to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase, which typically adds nucleotides to the 3' end of the nucleic acid.

[0034] 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 the same or have the same certain percentage of nucleotides, when compared or aligned for maximum correspondence as determined, for example, 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, see, 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, each of which is incorporated herein by reference.

[0035] "Modified nucleotide" in the context of oligonucleotides refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide that provides desired properties 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-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolo-pyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylriboU, 2'-0-methylriboC, N4-ethyl-dC, and N6-methyl-dA, etc. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or known in the art. In some embodiments, modified nucleotide substitutions modify the melting temperature (Tm) of oligonucleotides compared to the melting temperature of corresponding unmodified oligonucleotides. To further explain, some modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer-dimer formation, etc.), increase the yield of intended target amplicons, etc. 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.

[0036] A "variant" of a given oligonucleotide may contain one or more nucleotide additions, deletions or substitutions, for example, one or more nucleotide additions, deletions or substitutions at the 5'-end and / or 3'-end of the respective sequence of the oligonucleotide. 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. In that case, the probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modification, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or a phosphate-like moiety, or a combination thereof. For example, a "label" may be attached to the base portion of a "nucleotide", thereby resulting in a "modified nucleotide". A natural base in a "nucleotide" may also be replaced, for example, by 7-desazapurine, thereby resulting in a "modified nucleotide" as well. The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analog") differs from a naturally occurring nucleoside by some modification in the manner outlined above for "modified nucleotides" (or "nucleotide analogs").

[0037] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules, such as nucleic acid molecules encoding the nucleic acid sequence of the vanA gene or vanB gene, can be designed using computer programs such as, for example, OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Important features when designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size of the amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of the pair of primers, and the length of each primer (i.e., the primers must be long enough to anneal with sequence specificity and initiate synthesis, but not so long that fidelity is reduced during oligonucleotide synthesis). Typically, oligonucleotide primers are 8-50 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).

[0038] In addition to a set of primers, the method can use one or more probes to detect the presence or absence of vanA and / or vanB genes. The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that contains a specific nucleotide sequence that allows it to hybridize specifically (i.e., preferentially) to a "target nucleic acid" under a defined, given stringency by design or selection, in this case vanA (target) nucleic acid and / or vanB (target) nucleic acid. A "probe" can also be called a "detection probe", which means to detect a target nucleic acid.

[0039] In some embodiments, the described probes can be labeled with at least one fluorescent label. In one embodiment, the probes can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor fluorescent moiety, e.g., a quencher.

[0040] The design of oligonucleotides used as probes can be carried out in a similar manner to the design of primers. The embodiment can use a single probe or a pair of probes for the detection of amplification products. Depending on the embodiment, the use of one or more probes can include at least one label and / or at least one quencher moiety. As with primers, the probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long that it reduces accuracy during synthesis. Oligonucleotide probes are generally 15 to 30 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0041] The constructs can include vectors each containing one of the vanA or vanB primer and probe nucleic acid molecules (e.g., SEQ ID NOs: 1-15). The constructs can be used, for example, as control template nucleic acid molecules. Vectors suitable for use are commercially available and / or generated by recombinant nucleic acid techniques routine in the art. The vanA and vanB nucleic acid molecules can be obtained, for example, by chemical synthesis, by direct cloning from the vanA and vanB genes, or by PCR amplification.

[0042] Constructs suitable for use in the present methods typically include, in addition to vanA and vanB nucleic acid molecules (e.g., nucleic acid molecules containing one or more of the sequences of SEQ ID NOs: 1-15), a sequence encoding a selection marker (e.g., an antibiotic resistance gene) for selecting the desired construct and / or transformant, and an origin of replication. The choice of vector system usually depends on several factors, including, but not limited to, the choice of host cell, replication efficiency, selectability, inducibility, and ease of recovery.

[0043] Constructs containing vanA and vanB nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is meant to include prokaryotic and eukaryotic organisms, such as yeast, plant and animal cells. Prokaryotic hosts can include Escherichia coli, Salmonella typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis and tobacco. Constructs can be introduced into host cells using any of the techniques commonly known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and viral-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. In addition, naked DNA can be delivered directly to cells (see, eg, US Pat. Nos. 5,580,859 and 5,589,466).

[0044] Polymerase chain reaction (PCR) Conventional PCR techniques are disclosed in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188. 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 initiation 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 produced synthetically. Primers are preferably single-stranded for maximum efficiency in amplification, but may be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0045] 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 denaturing method, including physical, chemical, or enzymatic means. One method of separating the nucleic acid strands involves heating until the nucleic acid 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 will depend, for example, on the buffer salt concentration, and the length and nucleotide composition of the nucleic acid to be 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).

[0046] When the double-stranded template nucleic acid is denatured by heat, the reaction mixture can be cooled to a temperature that promotes the annealing of each primer to its target sequence on the described nucleic acid molecule. The annealing temperature is usually about 35°C to about 65°C, (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes the activity of the polymerase, i.e., a temperature sufficient for extension to occur from the annealed primers to generate products complementary to the template nucleic acid. The temperature must be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., temperatures for extension generally range from about 40° C. to about 80° C. (e.g., about 50° C. to about 70° C., about 60° C.)). Extension times 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).

[0047] PCR assays can use vanA and vanB gene nucleic acids, such as RNA or DNA (cDNA). The template nucleic acid need not be purified; it can be only a small portion of a complex mixture of vanA and / or vanB nucleic acids contained in a biological sample. VanA / vanB nucleic acid molecules can be extracted from biological samples by conventional techniques, such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids can be obtained from any number of sources, including plasmids, or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0048] The oligonucleotide primers are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 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. The reaction typically contains 150 to 320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0049] 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 many times as necessary to generate a desired amount of amplification product corresponding to the target nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in 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 will depend, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence enough for detection. Generally, the cycling steps are repeated at least about 20 times, but can be repeated 40, 60, or even 100 times.

[0050] Fluorescence Resonance Energy Transfer (FRET) FRET technology (e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance from each other, a visualized or otherwise detectable and / or quantifiable energy transfer occurs between the two fluorescent moieties. The donor typically transfers energy to the acceptor when excited by light irradiation of an appropriate wavelength. The acceptor typically re-emits the transferred energy in the form of light irradiation of a different wavelength. In some systems, non-fluorescent energy can be transferred between the donor and acceptor moieties by biomolecules that include a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0051] In one example, an oligonucleotide probe can contain a donor fluorescent moiety and a corresponding quencher, which may or may not be fluorescent, that dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the two fluorescent moieties, resulting in quenching of the fluorescence emission from the donor fluorescent moiety. 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. TM (BHQ)(Biosearch Technologies, Inc., Novato, Cal.), Iowa Black TM, (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry® Quencher 650 (BBQ-650), (Berry & Assoc., Dexter, Mich.).

[0052] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the target nucleic acid sequence. When the oligonucleotide probe hybridizes to the nucleic acid of the amplification product 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.

[0053] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (including appropriate dichroic mirrors and filters for monitoring fluorescence emission in a particular range), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed using an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation of the desired range.

[0054] As used herein with respect to a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, "corresponding" refers to an acceptor fluorescent moiety that has 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, so that efficient non-irradiative energy transfer can occur between them.

[0055] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) high efficiency Forster energy transfer; (b) large final Stokes shift (>100 nm); (c) as much shift of emission to the red portion of the visible spectrum (>600 nm); and (d) shift of emission to a wavelength higher than the Raman water fluorescence emission caused by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its excitation maximum near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0056] Representative donor fluorescent moieties that can be used in FRET technology with a variety of 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. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriaminepentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0057] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm is important since it affects the distance between the donor and acceptor fluorescent moieties. 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 from 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 methods for attaching the fluorescent moiety to the linker arm.

[0058] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to generate, for example, LC Red 640-labeled oligonucleotides. Linkers frequently used to couple donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (derived from fluorescein-NHS-ester, e.g., CX-Fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPG, which requires coupling of the fluorescein-NHS-ester after oligonucleotide synthesis.

[0059] Detection of vanA and vanB resistance genes The present disclosure provides a method for detecting the presence or absence of vanA and / or vanB genes in a biological or non-biological sample. The provided method avoids the problems of sample contamination, false negatives, and false positives. The method includes performing at least one cycling step including amplifying a portion of a vanA and / or vanB target nucleic acid molecule from a sample using multiple pairs of vanA and / or vanB primers, and a FRET detection step. The multiple cycling steps are preferably performed in a thermocycler. The method can be performed using vanA and vanB primers and probes to detect the presence of vanA and / or vanB genes, and detection of vanA and / or vanB in the assay indicates the presence of vanA and / or vanB in the sample.

[0060] As described herein, the 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 a single-stranded hybridization probe labeled with a fluorescent dye and a quencher, which may or may not be fluorescent, for example. When the first fluorescent moiety is excited with light of an appropriate wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is typically 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 degraded during the subsequent extension phase, for example by the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent 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 carrying out the methods described herein for detecting the presence or absence of vanA and / or vanB in a sample.

[0061] Molecular beacons in combination with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and a fluorescent label is typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows for secondary structure formation (e.g., a hairpin). As a result of the formation of secondary structures within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. 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, so that the emission of the first fluorescent moiety can be detected after excitation with light of an appropriate wavelength.

[0062] 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, e.g., fluorescein, is excited at 470 nm by the light source of the LightCycler® Instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, e.g., LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red 705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and when 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 to the number of original target DNA molecules. When amplification of the target nucleic acid occurs and the amplification product is purified, the hybridizing step results in a detectable FRET-based signal between the members of the probe pair.

[0063] Generally, the presence of FRET indicates the presence of target sequence in the sample, and the absence of FRET indicates the absence of target sequence in the sample.However, insufficient specimen collection, delayed transportation, improper transportation conditions, or the use of some 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 vanA and / or vanB infection.

[0064] Representative biological samples that can be used to carry out the method include, but are not limited to, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. The biological sample can be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release target gene nucleic acid, or in some cases, the biological sample can be directly contacted with PCR reaction components and appropriate oligonucleotides.

[0065] Melting curve analysis is an additional step that can be included in the cycling profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), defined as the temperature at which half of a DNA duplex separates into single strands. The melting temperature of DNA depends mainly on its nucleotide composition. Thus, 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, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature of one or more of the probes from the amplification product can confirm the presence or absence of the target sequence in the sample.

[0066] During each thermocycler run, control samples can be cycled as well. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the described target gene), for example, using a control primer and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing a target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., within the sample) or in a separate sample run alongside the patient sample, using the same primers and probes used to detect 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 a negative control, for example, lacking target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false positive signals. Thus, the control reaction can easily determine, for example, the ability of primers to anneal in a sequence-specific manner to initiate extension, and the ability of probes to hybridize in a sequence-specific manner to allow FRET to occur.

[0067] In one embodiment, the method includes a step to avoid contamination, for example, an enzymatic method utilizing uracil-DNA glycosylase is 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.

[0068] The method can be carried out using conventional PCR techniques combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714 and WO 97 / 46712.

[0069] The LightCycler® can be operated using a PC workstation and utilizes the Window NT operating system. Signals from samples are obtained as the machine sequentially positions the capillaries over the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The data generated can be stored for further analysis.

[0070] As an alternative to FRET, double-stranded DNA binding dyes such as fluorescent DNA binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)) can be used to detect the amplification products. Upon interaction with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent 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 double-stranded DNA binding dyes are used, a melting curve analysis is usually performed to confirm the presence of the amplification products.

[0071] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0072] Manufactured products / kits The present disclosure further provides an article of manufacture or kit for detecting vanA and vanB genes. The article of manufacture can include primers and probes used to detect vanA and / or vanB, along with suitable packaging materials. Representative primers and probes for detection of vanA and / or vanB can hybridize to vanA and / or vanB target nucleic acid molecules. In addition, the kit can also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to mecA / mecC-MRSA target nucleic acid molecules are provided.

[0073] The article of manufacture can include one or more fluorescent moieties for labeling the probes, or the probes provided with the kit can be labeled. For example, the article of manufacture can include donor and / or acceptor fluorescent moieties for labeling the vanA and vanB probes. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0074] The article of manufacture may also include a package insert or package label with instructions for detecting vanA and / or vanB in a sample using the vanA and / or vanB primers and probes. The article of manufacture may additionally include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents to prevent contamination) for carrying out the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0075] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. EXAMPLES

[0076] The following examples and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0077] Example I [Table 1-1] [Table 1-2] Table I. vanA / vanB primers and probes

[0078] vanA and vanB primer and probe sequences Table I shows the primers and probes used in the PCR assays for detection of the vanA and vanB genes.

[0079] Example 2 PCR experimental conditions Real-time PCR detection of vanA or vanB target genes was performed using the cobas® 4800 system or cobas® 6800 / 8800 system platform (Roche Molecular Systems, Inc., Pleasanton, Calif.). The final concentrations of amplification reagents were as follows:

[0080] Table II PCR Amplification Reagents [Table 2]

[0081] The table below shows a typical thermal profile used in a PCR amplification reaction.

[0082] Table III PCR Thermal Profile [Table 3]

[0083] The Pre-PCR program included incubations at 55°C, 60°C and 65°C for initial denaturation and reverse transcription of the RNA template. The incubation at the three temperatures compounds the advantageous effect that at the lower temperatures even slightly mismatched target sequences (such as genetic variants of the organism) are transcribed, while at the higher temperatures the formation of RNA secondary structures is suppressed, thus resulting in more efficient transcription. The PCR cycling is divided into two runs, both of which apply a one-stage setting (combining annealing and extension). The first 5 cycles at 55°C allow comprehensive gain by preamplifying the slightly mismatched target sequences, while the 45 cycles of the second run increase the specificity by using an annealing / extension temperature of 58°C.

[0084] Example 3 Performance of vanA and vanB primers and probes in PCR assays PCR experiments using non-benzylated or benzylated primers for the amplification of vanA and vanB genes in various strains of S. aureus, Enterococcus faecalis, and Enterococcus faecium were followed by detection using vanA or vanB gene probes. Figure 1 shows the results using non-benzylated primers SEQ ID NO: 1 and 3 in vanA MMx-1 column with detection by vanA probe SEQ ID NO: 5; SEQ ID NO: 6 and 8 in vanB MMx-1 column with detection by vanB probe SEQ ID NO: 10; and SEQ ID NO: 11 and 13 in vanB MMx-2 column with detection by vanB probe SEQ ID NO: 15. The presence and detection of each amplification product is shown as Ct values. Figure 2 shows the results of using benzylated primers SEQ ID NOs: 2 and 4 in the vanA MMx-1 column with detection by the vanA probe of SEQ ID NO: 5, SEQ ID NOs: 7 and 9 in the vanB MMx-1 column with detection by the vanB probe of SEQ ID NO: 10, and SEQ ID NOs: 12 and 14 in the vanB MMx-2 column with detection by the vanB probe of SEQ ID NO: 15. The presence and detection of each amplification product is shown as Ct values.

[0085] No significant difference in performance was observed between benzylated and non-benzylated primers, and more importantly, the pair of vanA primers and probes and the two pairs of vanB primers and probes were all able to amplify and detect their respective target nucleic acids in a specific manner.

[0086] Example 4 Multiplex PCR assay for the detection of VRSA and MRSA. A multiplex PCR assay was set up for detection of both vancomycin-resistant S. aureus (VRSA) by targeting the vanA and vanB genes, and methicillin-resistant S. aureus (MRSA) by targeting the mecA and mecC genes, as well as the SCCmec OrfX right extremity junction (OrfX). The primers and probes for this assay are shown in Table IV. The results of the PCR assay using these primer and probe sets on various S. aureus and Enterococcus strains are shown in Figure 3.

[0087] TABLE IV Primers and probes for detecting VRSA and MRSA [Table 4] [Table 5]

Claims

1. 1. A method for detecting bacteria having the vanA gene and / or the vanB gene in a sample, comprising: - if said vanA gene and / or vanB gene are present in the sample, performing an amplification step comprising contacting said sample with a vanA forward and reverse primer set and a vanB forward and reverse primer set to generate amplification products; - carrying out a hybridization step comprising contacting said amplification products with one or more detectable vanA probes and one or more detectable vanB probes; and - detecting the presence or absence of said amplification product, wherein the presence of said amplification product indicates the presence of said vanA and / or vanB genes in said sample and the absence of said amplification product indicates the absence of said vanA and / or vanB genes in said sample; The method comprising: the vanA primer set comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 1 or 2, and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 3 or 4; the vanB primer set comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 6, 7, 11, and 12, and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 8, 9, 13, and 14; and the one or more detectable vanA probes comprise the oligonucleotide sequence of SEQ ID NO: 5 or its complement, and the one or more detectable vanB probes comprise the oligonucleotide sequence of SEQ ID NO: 10 or 15 or its complement; The method.

2. the hybridizing step comprises contacting the amplification product with a detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; and 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, the presence or absence of fluorescence FRET indicating the presence or absence in the sample. The method of claim 1.

3. The method of claim 1 or 2, wherein the amplification step uses a polymerase enzyme having 5' to 3' nuclease activity.

4. 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.

5. The vanA primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:2 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:4; and The vanB primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 7 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 9; and the one or more detectable vanA probes comprise the oligonucleotide sequence of SEQ ID NO:5, and the one or more detectable vanB probes comprise the oligonucleotide sequence of SEQ ID NO:10; 3. The method according to claim 1 or 2.

6. A primer and probe set for amplifying and detecting a vanA gene target sequence, comprising: at least one forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 1 or 2, - at least one reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 3 or 4, and at least one detectable probe for the detection of vanA amplification products comprising the oligonucleotide sequence of SEQ ID NO: 5 or its complement; A primer and probe set comprising:

7. The primer and probe set of claim 6 , wherein the detectable probe comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.

8. A primer and probe set for amplifying and detecting a vanB gene target sequence, comprising: - at least one forward primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 6, 7, 11 and 12, - at least one reverse primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 8, 9, 13 and 14, and - at least one detectable probe for the detection of vanB amplification products comprising an oligonucleotide sequence selected from SEQ ID NOs: 10 and 15 or their complements; A primer and probe set comprising:

9. 9. The primer and probe set of claim 8, wherein the detectable probe comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.

10. A kit for detecting nucleic acids of vanA-containing Staphylococcus aureus, comprising the set of primers and probes described in claim 6 or 7, and further comprising at least one of nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase.

11. A kit for detecting nucleic acids of vanB-containing Staphylococcus aureus, comprising the set of primers and probes described in claim 8 or 9, and further comprising at least one of nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase.

12. A kit for detecting nucleic acids of vanA-containing and / or vanB-containing Staphylococcus aureus, comprising a primer and probe set according to claim 6 or 7 and a primer and probe set according to claim 8 or 9, and further comprising at least one of nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase.

13. 1. A method for the detection of vancomycin-resistant (VRSA), methicillin-resistant (MRSA), or both VRSA and MRSA Staphylococcus aureus (S. aureus), comprising: The method comprises: - carrying out an amplification step, contacting the sample with a set of vanA primers comprising at least a forward and a reverse primer and a set of vanB primers comprising at least a forward and a reverse primer to produce amplification products if vanA and / or vanB genes are present in the sample; contacting the sample with a set of CPE primers comprising at least a forward and a reverse primer to generate an amplification product of S. aureus capsular polysaccharide enzyme (CPE) if S. aureus is present in the sample; contacting the sample with a set of Staphylococcal chromosomal cassette mec (SCCmec) primers comprising at least a forward and a reverse primer to generate an amplification product of the right end junction of the SCCmec mobile genetic element, if SCCmec is present in the sample; and contacting the sample with a set of mecA primers comprising at least a forward and a reverse primer and a set of mecC primers comprising at least a forward and a reverse primer to produce amplification products if mecA and / or mecC genes are present in the sample; performing the amplification step, - carrying out a hybridization step comprising contacting said amplification products with one or more detectable vanA probes, one or more detectable vanB probes, one or more detectable CPE probes, one or more detectable SCCmec probes, and one or more detectable mecA probes and one or more detectable mecC probes; and - detecting the presence or absence of said amplification products, wherein the presence of amplification products of the vanA and / or vanB genes and the CPE gene indicates the presence of VRSA in said sample, and the presence of amplification products of the SCCmec element, mecA and / or mecC genes and the CPE gene indicates the presence of MRSA in said sample. Including, A method in which the vanA primer and probe set comprises the primer and probe set of claim 6 or 7, and / or the vanB primer and probe set comprises the primer and probe set of claim 8 or 9.

14. the vanA primer set comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 1 or 2 and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 3 or 4, and the one or more detectable vanA probes comprise the oligonucleotide sequence of SEQ ID NO: 5 or its complement; the set of vanB primers comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 6, 7, 11, and 12, and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 8, 9, 13, and 14, and the one or more detectable vanB probes comprise the oligonucleotide sequence of SEQ ID NO: 10 or 15, or a complement thereof; the set of CPE primers comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 16 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 17, and the one or more detectable CPE probes comprise the oligonucleotide sequence of SEQ ID NO: 18 or its complement; the set of SCCmec primers comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 19 and 20 and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 21, 22, 23, and 24, and the one or more detectable vanB probes comprise the oligonucleotide sequence of SEQ ID NO: 25 or its complement; the mecA primer set comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:26 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:27, and the one or more detectable mecA probes comprise the oligonucleotide sequence of SEQ ID NO:28 or its complement; and the set of mecC primers comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:29 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:30, and the one or more detectable mecC probes comprise the oligonucleotide sequence of SEQ ID NO:31 or its complement; The method of claim 13.

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