Patent ID: 7297517

Claim:
A method for detecting methicillin-resisant Staphylococcus aureus (MRSA) in a sample, said method comprising the steps of: (a) preparing a reaction mcomprising: a sample; a first oligonucleotide primer comprising (i) a portion of the mecA gene of MRSA, wherein said portion is a target sequence and (ii) an RNA polymerase promoter sequence attached to the 5′-end of the sequence in (i); a second oligonucleotide primer; an enzyme or a mixture of enzymes having (i) RNA-dependent DNA polymerase activity, (ii) ribonuclease activity that hydrolyzes RNA of an RNA-DNA hybrid without hydrolyzing single-stranded and double-stranded RNA or DNA, (iii) DNA-dependent DNA polymerase activity, and (iv) DNA-dependent RNA polymerase activity; and a cleaving oligonucleotide probe comprising a sequence complementary to a region overlapping with and adjacent to said target sequence; (b) incubating said reaction mixture under conditions that allow the formation of a double-stranded cDNA product from the target sequence and the transcription of an RNA product from the double-stranded cDNA product; and (c) detecting the RNA product transcribed from the double-stranded cDNA product,, wherein: (1) an oligonucleotide comprising at least 10 contiguous bases of the sequence recited in SEQ ID No:18 is used as the first primer, an oligonucleotide comprising least 10 contiguous bases of the sequence recited in any of SEQ ID No:19, 20 or 21 is used as the second primer, and an oligonucleotide comprising the sequence recited in SEQ ID No:26 is used as the cleaving probe, or (2) an oligonucleotide comprising at least 10 contiguous bases of the sequence recited in SEQ ID No:22 is used as the first primer, an oligonucleotide comprising at least 10 contiguous bases of the sequence recited in any of SEQ ID No:23 or 24 is used as the second primer, and an oligonucleotide comprising the sequence recited in SEQ ID No:27 is used as the cleaving probe, or (3) an oligonucleotide comprising at least 10 contiguous bases of the sequence recited in SEQ ID No:25 is used as the first primer, an oligonucleotide comprising at least 10 contiguous bases of the sequence recited in any of SEQ ID No:23 or 24 is used as the second primer, and an oligonucleotide comprising the sequence recited in SEQ ID No:28 is used as the cleaving probe.