Detection of methicillin-resistant staphylococcus aureus

Abstract

The present invention provides improved tests for the detection of methicillin-resistant Staphylococcus aureus . The tests are particularly useful for eliminating false positive results due to the presence of a mixed bacterial population in patient samples.

1 - 47 . (canceled) 
     
     
         48 . A method of identifying the presence in a sample of a methicillin-resistant  Staphylococcus aureus  (MRSA) having an insertion of an SCCmec cassette within  Staphylococcus aureus  chromosomal DNA which comprises the steps of:
 bringing, in a single container, the biological sample in contact with
 a) a first oligonucleotide set comprising
 (1) a first oligonucleotide having a nucleic acid sequence that specifically hybridizes to an extremity junction region of a SCCmec cassette, and 
 (2) a second oligonucleotide having a nucleic acid sequence that specifically hybridizes to a  Staphylococcus aureus  chromosomal DNA region flanking said SCCmec cassette to form a first reaction product of the biological sample and the first and second oligonucleotides, and 
 
 b) a second oligonucleotide set comprising
 (3) a third oligonucleotide having a nucleotide sequence that specifically hybridizes to a first region of mecA nucleic acid and 
 (4) a fourth oligonucleotide having a nucleotide sequence that specifically hybridizes to a second region of mecA nucleic acid to form a second reaction product of the biological sample and the third and fourth oligonucleotides; and 
 
   identifying the presence of MRSA by detecting both a first and a second reaction product.   
     
     
         49 . The method of  claim 48 , wherein the contacting step further comprises performing an amplification reaction using oligonucleotide sets (a) and (b) as primers, and wherein said identifying step comprises detecting the presence or absence of an amplification product from both oligonucleotide sets. 
     
     
         50 . The method of  claim 49 , wherein the identifying step comprises contacting the first and second reaction products with (1) a first probe capable of specifically hybridizing with the first reaction product and (2) a second probe capable of specifically hybridizing with the second reaction product. 
     
     
         51 . The method of  claim 48 , wherein the first oligonucleotide comprises more than one oligonucleotide capable of hybridizing in an extremity junction region of the SCCmec cassette. 
     
     
         52 . The method of  claim 50 , wherein the first probe comprises more than one probe, each capable of hybridizing specifically to a different type of MRSA. 
     
     
         53 . The method of  claim 50 , wherein the first probe comprises five or more probes, each capable of hybridizing specifically to a different type of MRSA. 
     
     
         54 . The method of  claim 48 , wherein the extremity junction region is the right extremity junction region. 
     
     
         55 . The method of  claim 54 , wherein the second oligonucleotide specifically hybridizes to a region of orfX. 
     
     
         56 . The method of  claim 55 , wherein the second oligonucleotide comprises the nucleic acid set forth in SEQ ID NO: 13. 
     
     
         57 . The method of  claim 48 , wherein the third oligonucleotide comprises a nucleic acid selected from the group consisting of SEQ ID NO: 15 and 16. 
     
     
         58 . The method of  claim 48 , wherein the fourth oligonucleotide comprises a nucleic acid selected from the group consisting of SEQ ID NO: 15 and 16. 
     
     
         59 . The method of  claim 50 , wherein the first probe comprises a sequence selected from the group consisting of: SEQ ID NO: 7, 8, 9, 10, 11, and 12. 
     
     
         60 . The method of  claim 50 , wherein detection is performed using, as the first probe, at least five nucleic acids, each comprising one of SEQ ID NO: 7, 8, 9, 10, 11, and 12. 
     
     
         61 . The method of  claim 49 , wherein amplification is performed using, as the first oligonucleotide, a nucleic acid comprising a sequence selected from the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5, and 6. 
     
     
         62 . The method of  claim 49 , wherein amplification is performed using as the first oligonucleotide at least five nucleic acids, each comprising one of SEQ ID NO: 1, 2, 3, 4, 5, and 6. 
     
     
         63 . The method of  claim 49 , wherein the amplification reaction utilizes a mecA oligonucleotide comprising a nucleic acid selected from the group consisting of SEQ ID NO: 15 and 16. 
     
     
         64 . The method of  claim 50 , wherein detection of the second reaction product utilizes a mecA probe comprising the nucleic acid set forth in SEQ ID NO: 14.