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A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing

Lin Cai^1,, Fanrong Kong^2,, Qinning Wang², Huiping Wang³, Meng Xiao⁴, Vitali Sintchenko^2,5 and Gwendolyn L. Gilbert^2,5

¹ Department of Dermatology, Peking University People's Hospital, Beijing 100044, PR China

² Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia

³ Department of Dermatology, Tianjin Medical University General Hospital, Tianjin, PR China

⁴ Life Science College, Peking University, Beijing, PR China

⁵ Western Clinical School, University of Sydney, Sydney, Australia

Correspondence
Gwendolyn L. Gilbert
l.gilbert{at}usyd.edu.au

Received November 12, 2008
Accepted May 5, 2009

The aim of this study was to develop a new discriminatory method for MRSA SCCmec typing based on multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay to enable rapid identification and classification of MRSA SCCmec types in a clinical laboratory. Forty-five primer sets and 49 probes were designed and tested in uniplex PCR (uPCR) and mPCR/RLB. Probes were compared in silico to 14 whole-genome sequences and 18 partial SCCmec gene sequences of Staphylococcus aureus and complete genome and partial SCCmec genes of seven non-MRSA strains, including meticillin-susceptible S. aureus and meticillin-resistant coagulase-negative staphylococci. The method was tested on a set of 42 well-characterized reference MRSA strains. It identified all five epidemiologically relevant SCCmec types and 26 subtypes, including established and new subtypes of SCCmec III, IV (eight subtypes each) and V (three subtypes). The discriminatory power of mPCR/RLB SCCmec typing was similar to that of MLST and spa typing (Simpson indices of diversity of 0.916, 0.926 and 0.882, respectively; differences not statistically significant). The application of mPCR/RLB hybridization assay to MRSA SCCmec typing can improve the specificity, discriminatory power and throughput of the typing procedure. The detection of up to 43 mPCR products in a single hybridization assay transforms MRSA SCCmec typing from passive epidemiological library typing into a potential tool for near-real-time infection control surveillance and tracking of MRSA transmission in hospitals.

These authors contributed equally to this work.