New Real-Time PCR Assay for Rapid Detection of Methicillin- ResistantStaphylococcus aureusDirectly from Specimens Containing a Mixture of Staphylococci

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Abstract
Molecular methods for the rapid identification of methicillin-resistantStaphylococcus aureus(MRSA) are generally based on the detection of anS. aureus-specific gene target and themecAgene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) andS. aureus, either of which can carrymecA.In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in mec(SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to theS. aureuschromosomalorfXgene sequences located to the right of the SCCmecintegration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptibleS. aureus(MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was ∼25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.

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