Detection of methicillin-resistant staphylococcus aureus in biological samples

A method for determining the presence or absence of methicillin resistant Staphylococcus aureus (MRSA) in a biological sample comprising:

• (a) contacting the biological sample with:
• (i) a first primer pair that specifically hybridizes under stringent conditions to a segment of a target nucleic acid specific for Staphylococcus aureus, if present,
• (ii) a second primer pair that specifically hybridizes under stringent conditions to a segment of a target mecA nucleic acid if present, and
• (iii) a third primer pair that specifically hybridizes under stringent conditions to a segment of a target mecC nucleic acid, if present,
• to produce a reaction-sample mixture, wherein at least one primer of each primer pair is associated with a fluorophore label,
• (b) subjecting the reaction-sample mixture to real-time polymerase chain reaction (PCR) conditions under which each of the target nucleic acids present in the biological sample is amplified to produce a fluorescent signal,
• (c) measuring the amount of fluorescent signal produced by each fluorophore, and
• (d) determining the presence or absence of MRSA by comparing the amount of fluorescence from the target marker nucleic acids, whereby
• (i) MRSA is determined to be present in the biological sample when a fluorescent signal is detected for both the S. aureus specific target nucleic acid and the mecA and/or mecC nucleic acids and
• (1) the cycle threshold (Ct) from the S. aureus specific target nucleic acid minus the Ct from the mecA and/or mecC nucleic acids≦1.9, or
• (2) the Ct from the S. aureus specific target nucleic acid minus the Ct from the mecA and/or mecC nucleic acids>1.9 and the Ct from the mecA and/or mecC nucleic acids plus 1.9<Ct from the S. aureus specific target nucleic acid;
• (ii) S. aureus and a methicillin resistance gene are determined to be present in the biological sample when a Ct is detected for both the S. aureus specific target nucleic acid and the mecA and/or mecC nucleic acids, and the Ct from the S. aureus specific target nucleic acid minus the Ct from the mecA and/or mecC nucleic acids<1.9, and the Ct from the S. aureus specific target nucleic acid plus 1.9<the Ct from the mecA and/or mecC nucleic acids;
• or
• (iii) S. aureus is determined to be present and MRSA is determined to be absent in the biological sample if a fluorescent signal is detected for the S. aureus specific target nucleic acid sequence but no fluorescent signal is detected for mecA and/or mecC.