Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus

Abstract

Staphylococcal cassette chromosome mec (SCCmec) typing is essential for understanding the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA). SCCmec elements are currently classified into types I to V based on the nature of the mec and ccr gene complexes, and are further classified into subtypes according to their junkyard region DNA segments. Previously described traditional SCCmec PCR typing schemes require multiple primer sets and PCR experiments, while a previously published multiplex PCR assay is limited in its ability to detect recently discovered types and subtypes such as SCCmec type V and subtypes IVa, b, c, and d. We designed new sets of SCCmec type- and subtype-unique and specific primers and developed a novel multiplex PCR assay allowing for concomitant detection of the methicillin resistance (mecA gene) (also serving as an internal control) to facilitate detection and classification of all currently described SCCmec types and subtypes I, II, III, IVa, b, c, d, and V. Our assay demonstrated 100% sensitivity and specificity in accurately characterizing 54 MRSA strains belonging to the various known SCCmec types and subtypes, when compared with previously described typing methods. Further application of our assay in 453 randomly selected local clinical isolates confirmed its feasibility and practicality. This novel assay offers a rapid, simple, and feasible method for SCCmec typing of MRSA, and may serve as a useful tool for clinicians and epidemiologists in their efforts to prevent and control infections caused by this organism.

FIG. 1.

New multiplex PCR assay identifies SCCmec types and subtypes I, II, III, IVa, IVb, IVc, IVd, and V, and simultaneously detects the methicillin resistance (mecA gene). Type I, lanes 1 to 3 (strains NCTC10442, COL, and PER34, respectively); type II, lanes 4 to 6 (strains N315, CLS-5153, and CLS-440, respectively); type III, lanes 7 to 9 (strains 85/2082, ANS46, and CMRSA-3, respectively); type IVa, lanes 10 to 12 (strains CA05, N02-590, and CLS-2207, respectively); type IVb, lanes 13 to 15 (strains 8/6-3P, CLS-4584, and CLS-5827, respectively); type IVc, lanes 16 and 17 (strains MR108 and CLS-1040, respectively); type IVd, lanes 18 and 19 (strains JCSC4469 and CMRSA-5, respectively); type V, lane 20 (strain WIS [WBG8318]-JCSC3624); lane 21, negative control; and lanes M, molecular size markers, 100-bp DNA ladder (BioLabs). Refer to Table 3 for details of each strain.