Objectives: To develop and validate a multiplex PCR-based reverse line blot hybridization (mPCR/RLB) strategy for subtyping staphylococcal cassette chromosome mec (SCCmec) type IV methicillin-resistant Staphylococcus aureus (MRSA) strains. Methods: Sixty primer pairs and 63 probes were designed on the basis of the available sequences of each open-reading frame (ORF) at GenBank, for the previously described SCCmec IV subtypes. Probes were compared in silico to 18 whole genome sequences complete

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... enome and partial SCCmec gene of 6 non-MRSA strains, including methicillin susceptible S. aureus and methicillin resistant coagulase negative staphylococci. Results: The mPCR/RLB assay classified a set of 93 MRSA strains which possessed SCCmec type IV into 68 subtypes, including 46 subtypes for 52 well-characterized reference strains and 22 subtypes for 41 clinical strains. The discriminatory power (Simpson indices of diversity of 0. 987) is higher than other genotyping methods to date. It can rapidly and simultaneously detect 43 samples with a turn-around time (including culturing of isolates, DNA extraction, mPCR setup and running, and RLB hybridization) of about two working days. This comparative analysis the potential sensitivity of probes demonstrated that each of the 63 probes found homologous match with at least one GenBank MRSA SCCmec type IV sequence. Conclusion: The application of mPCR/RLB hybridization assay to MRSA SCCmec IV subtyping can improve the specificity, discriminatory power and throughput of the typing procedure. Moreover it is also a good tool for near-real time infection control surveillance and MRSA transmission tracking.