title: A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing.
abstract: 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.
authors.affiliation: Department of Dermatology, Peking University People's Hospital, Beijing 100044, PR China.
authors.forename: Lin
authors.initials: L
authors.lastname: Cai
authors.affiliation: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia.
authors.forename: Fanrong
authors.initials: F
authors.lastname: Kong
authors.affiliation: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia.
authors.forename: Qinning
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Dermatology, Tianjin Medical University General Hospital, Tianjin, PR China.
authors.forename: Huiping
authors.initials: H
authors.lastname: Wang
authors.affiliation: Life Science College, Peking University, Beijing, PR China.
authors.forename: Meng
authors.initials: M
authors.lastname: Xiao
authors.affiliation: Western Clinical School, University of Sydney, Sydney, Australia.|Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia.
authors.forename: Vitali
authors.initials: V
authors.lastname: Sintchenko
authors.affiliation: Western Clinical School, University of Sydney, Sydney, Australia.|Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia.
authors.forename: Gwendolyn L
authors.initials: GL
authors.lastname: Gilbert
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005838:Genotype; D008712:Methicillin / Q000494:pharmacology; D016106:Methicillin Resistance / Q000235:genetics; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D008712:Methicillin