Multiplex real-time PCR for rapid Staphylococcal cassette chromosome mec typing

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 (SCCmec) typing. The design of this system is based on the established definition of SCCmec 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 SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the DeltamecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the DeltamecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec 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 SCCmec typing of MRSA isolates.

FIG. 1.

Schematic representation of SCCmec types I to VI and recently described type VIII in MRSA prototype strains. SCCmec structures are illustrated according to nucleotide sequences deposited in GenBank under the following accession numbers (with strains and SCCmec types given in parentheses): AB033763 (NCTC10442, SCCmec I), D86934 (N315, SCCmec II), AB037671 (85/2082, SCCmec III), AB063172 (CA05, SCCmec IV), AB121219 (WIS, SCCmec V), AF411935 (HDE288, SCCmec VI), and FJ390057 (C10682, SCCmec VIII). The ccr and mec class complexes grey and utilized for SCCmec type classification are shaded and shown against a grey and green background, respectively; elements outside of these areas constitute “joining” regions (J1, J2, and J3, from right to left).

FIG. 2.

Stratagene Mx4000 real-time PCR amplification curves for SCCmec type I to VI prototype strains (NCTC10442, N315, 85/2082, CA05, WIS, and HDE288). (Left) MB-PCR panel I results; (right) MB-PCR panel II results. Each MB-PCR panel consists of four molecular beacon probes labeled with different fluorescent dyes, as indicated in the keys at the bottom.