Compositions and methods for detecting methicillin-resistant staphylococcus aureus

Abstract

Methods for the 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 are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers, probes targeting the genes for mecA-MRSA and mecC-5 MRSA, along with kits are provided that are designed for the detection of mecA/mecC-MRSA.

1 - 25 . (canceled) 
     
     
         26 . A method of detecting mecA-containing methicillin-resistant  Staphylococcus aureus  (mecA-MRSA) in a sample, the method comprising:
 performing an amplifying step comprising contacting the sample with a forward primer comprising a nucleotide sequence of SEQ ID NO: 1, or a complement thereof, and a reverse primer comprising a nucleotide sequence of SEQ ID NO: 2, or a complement thereof, to produce an amplification product if mecA-MRSA is present in the sample;   performing a hybridizing step comprising contacting the amplification product with one or more detectable mecA-MRSA probes wherein one of the one or more detectable mecA-MRSA probe comprises the sequence of SEQ ID NO: 3, or a complement thereof; and   detecting the presence or absence of the amplified amplification product, wherein the presence of the amplified amplification product is indicative of the presence of mecA-MRSA in the sample and wherein the absence of the amplified amplification product is indicative of the absence of mecA-MRSA in the sample.   
     
     
         27 . The method of  claim 26 , wherein:
 the hybridizing step comprises contacting the amplification product with a probe that is 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, wherein the presence or absence of fluorescence FRET is indicative of the presence or absence of mecA-MRSA in the sample.   
     
     
         28 . The method of  claim 27 , wherein the donor fluorescent moiety and the corresponding acceptor fluorescent moiety are within no more than 8 nucleotides of each other on the probe. 
     
     
         29 . The method of  claim 27 , wherein the acceptor fluorescent moiety is a quencher. 
     
     
         30 . The method of  claim 26 , wherein said amplifying step employs a polymerase enzyme having 5′ to 3′ nuclease activity. 
     
     
         31 . The method of  claim 26  further comprising detecting in the same reaction mecC-containing methicillin-resistant  Staphylococcus aureus  (mecC-MRSA) in said sample by
 further contacting the sample in the amplifying step with a forward primer comprising a nucleotide sequence of SEQ ID NO: 4 or 5, or a complement thereof, and a reverse primer comprising a nucleotide sequence of SEQ ID NO: 6, or a complement thereof, to produce an amplification product if mecC-MRSA is present in the sample; 
 further contacting the amplification product in the hybridizing step with one or more detectable mecC-MRSA probes wherein one of the one or more detectable mecC-MRSA probe comprises the sequence of SEQ ID NO: 7, or a complement thereof; and 
 detecting the presence or absence of the amplified amplification product, wherein the presence of the amplified amplification product is indicative of the presence of mecC-MRSA in the sample and wherein the absence of the amplified amplification product is indicative of the absence of mecC-MRSA in the sample. 
 
     
     
         32 . The method of  claim 31 , wherein the mecC-MRSA probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, which differs from the donor fluorescent moiety and the corresponding acceptor fluorescent moiety of the labeled mecA-MRSA probe. 
     
     
         33 . A method of detecting mecC-containing methicillin-resistant  Staphylococcus aureus  (mecC-MRSA) in a sample, the method comprising:
 performing an amplifying step comprising contacting the sample with a forward primer comprising a nucleotide sequence of SEQ ID NO: 4 or 5, or a complement thereof, and a reverse primer comprising a nucleotide sequence of SEQ ID NO: 6 to produce an amplification product if mecC-MRSA is present in the sample;   performing a hybridizing step comprising contacting the amplification product with one or more detectable mecC-MRSA probes wherein one of the one or more detectable mecC-MRSA probe comprises the sequence of SEQ ID NO: 7, or a complement thereof; and   detecting the presence or absence of the amplified amplification product, wherein the presence of the amplified amplification product is indicative of the presence of mecC-MRSA in the sample and wherein the absence of the amplified amplification product is indicative of the absence of mecC-MRSA in the sample.   
     
     
         34 . The method of  claim 33 , wherein:
 the hybridizing step comprises contacting the amplification product with a probe that is 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, wherein the presence or absence of fluorescence FRET is indicative of the presence or absence of mecC-MRSA in the sample.   
     
     
         35 . The method of  claim 34 , wherein the donor fluorescent moiety and the corresponding acceptor fluorescent moiety are within no more than 8 nucleotides of each other on the probe. 
     
     
         36 . The method of  claim 34 , wherein the acceptor fluorescent moiety is a quencher. 
     
     
         37 . The method of  claim 34 , wherein said amplifying step employs a polymerase enzyme having 5′ to 3′ nuclease activity. 
     
     
         38 . A kit for detecting a nucleic acid of mecA-containing  Staphylococcus aureus  (mecA-MRSA) comprising:
 a first oligonucleotide comprising a sequence of SEQ ID NO: 1, or a complement thereof;   a second oligonucleotide comprising a sequence of SEQ ID NO: 2, or a complement thereof; and   a third detectably labeled oligonucleotide configured to hybridize to an amplicon generated by the first oligonucleotide and the second oligonucleotide, comprising SEQ ID NO: 3, or a complement thereof.   
     
     
         39 . The kit of  claim 38 , wherein the third detectably labeled oligonucleotide comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, wherein the acceptor fluorescent moiety is a quencher. 
     
     
         40 . The kit of  claim 39  further comprising nucleoside triphosphates, nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase. 
     
     
         41 . The kit of  claim 39  for further detecting a nucleic acid of mecC-containing  Staphylococcus aureus  (mecC-MRSA), said kit further comprising:
 a fourth oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 4 and 5, or a complement thereof; 
 a fifth oligonucleotide comprising a sequence of SEQ ID NO: 6, or a complement thereof; and 
 a sixth detectably labeled oligonucleotide configured to hybridize to an amplicon generated by the forth oligonucleotide and the fifth oligonucleotide, comprising SEQ ID NO: 7, or a complement thereof. 
 
     
     
         42 . The kit of  claim 41 , wherein the sixth detectably labeled oligonucleotide probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, which differs from the donor fluorescent moiety and the corresponding acceptor fluorescent moiety of the third detectably labeled oligonucleotide. 
     
     
         43 . A kit for detecting a nucleic acid of mecC-containing  Staphylococcus aureus  (mecC-MRSA) comprising:
 a first oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 4 and 5, or a complement thereof;   a second oligonucleotide comprising a sequence of SEQ ID NO: 6, or a complement thereof; and   a third detectably labeled oligonucleotide configured to hybridize to an amplicon generated by the first oligonucleotide and the second oligonucleotide, comprising SEQ ID NO: 7, or a complement thereof.   
     
     
         44 . The kit of  claim 44 , wherein the third detectably labeled oligonucleotide comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, wherein the acceptor fluorescent moiety is a quencher. 
     
     
         45 . The kit of  claim 45 , further comprising nucleoside triphosphates, nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase.