Multiplex Real-Time PCR for Rapid Staphylococcal Cassette Chromosome mec Typing

Abstract

ABSTRACT Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCC mec ) typing. The design of this system is based on the established definition of SCC mec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets ( mec class , ccr ) for each SCC mec type. MB-PCR panel I targets mecA , ccrB2 , mecI , and the Δ mecR1- IS 1272 junction ( mec class B); it can definitively identify SCC mec types II and IV. MB-PCR panel II detects ccrC , ccrB1 , ccrB3 , ccrB4 , and the Δ mecR1- IS 431 junction ( mec class C2) and is therefore capable of identifying SCC mec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCC mec type VIII ( ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCC mec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital- and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCC mec typing of MRSA isolates.