Summary

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Published: September 05, 2013
doi:

Summary

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Abstract

??????????????? ??????? ????????? MEC (SCC MEC) ????????? ???????? ????? ?????? ???????????? ???????????? ??? ????????? ??????? ????????????? ? ??????????? ????? ??????????-?????????? ?????????? ??????????? (MRSA), ? ?????????, ? ?????? ????????? ????????????? MRSA (CA-MRSA), ???????????? ?? ????? ????. ???????????? ?????? ??? ????? ????????????? SCC ??? ????? ?????? ? ????????????? ?????????? ??? ? CCR ??? ??????? ?????, ?? ??????? ????????????? ????? ??????? ????????? ? ?????????? ????????? ????????????? ???. ?? ??????????? ? ???????????? ??????? ??????????? ???-??????? ??? ???????? ?????? ??????? SCC MEC ????? ? ???????? ? ? V, ? ????? ??????? ??? ??? ????? ?????????????? ?????????????????? ????? ?????????. ???? ??????? ?????? ????? ????????? SCC MEC ????? ? ????? ???????, ????? ???????????????? ? ?????? ??????????? ?? ???? ????. ?????? ??-?? ??????? ????? ?? ??????? ? ???????? ????? ?????????, ?????????????? ? ??????????? ?????????? ??????????? ????????? ??? ????????? ???? ??????? ????????? ???????????. ??? ?????????? ???????? ???????? MRSA SCC MEC ???????, ????? ?? ???????, ??? ??????? ??? ??????????? ???-???????, ? ????? ???????? ????????? ???????? ?????? ???? ? ??????????? ??????, ??????? ?????? ??? ????????.

Introduction

??????????-?????????? ?????????? ??????????? (MRSA) ????????? ? ??????????????? ? ????? 21-67-?? ????????? ???????????? ?????????, ?????????? ??????????????? ??????? ????????? MEC (SCC MEC), ??????? ???? ? ???? ???????????? ? ??????????? (M

Protocol

??? ????????? ?????? ??????????? ? ?????? ??????????????? 2 ????????????????? ????????????. ??????????????? ???????? ?????????????? ??????, ????? ??? ???????? ? ?????? ?????? ???? ???????????? ? ????? ?????. 1. ?????????? ???????? ?????? ?????? ???????? ??????? MRSA ?????????. ? ???? ????? ??? ?????? ???? ???????, ???????? ???? ??????? MRSA ?????????? ???????? ???????? ? ??????????? ??????? ?????? ???????? ?? ???????????? ?????? ???? (TSA) ????????. ????????? ???????? ????? ??????…

Representative Results

This updated M-PCR is able to determine (classify) SCCmec types I-V, as well major subtypes of SCCmec IV. The assay targets unique and specific loci of SCCmec I, II, III, IVa, IVb, IVc, IVd, IVE and V, with concomitant mecA gene detection. Tentative identification of SCCmec VI and VIII is also possible, but would require further testing to confirm. Figure 3 illustrates representative SCCmec I-VI and VIII and the locations of each target within the ele…

Discussion

The multiplex PCR assay described here provides a simple, reliable method for classifying types and major subtypes of SCCmec I-V in Staphylococci, with simultaneous discrimination of MRSA from MSSA. The assay is robust, conveniently done in a single PCR reaction and results are easy to interpret. The assay does have limitations in that it targets individual loci within each SCCmec type, but does not offer comprehensive ccr and mec complex typing, and therefore cannot be considered a go…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank T. Ito, K. Hiramatsu, R. Daum, H. de Lencastre, and D. Coleman for the kind gift of some reference strains used in this study. This work was in part supported financially by the Centre for Antimicrobial Resistance (CAR), Alberta Health Services-Calgary/Calgary Laboratory Services/University of Calgary.

Materials

Name of Reagent Company Catalogue Number
Tryptic Soy Agar Fisher (BD) CA90002-700 (236920)
Sterile distilled water Life Technologies 10977-015
Platinum Taq: including 10x PCR buffer and 50 mM MgCl2 Life Technologies 10966-034
100 mM dNTP Life Technologies 10297-018
Tris base Sigma-Aldrich T6066
Boric acid Sigma-Aldrich B7901
EDTA Life Technologies 15576-028
Agarose Life Technologies 16500-500
Glycerol Life Technologies 15514-011
Bromophenol blue Sigma-Aldrich B8026
1Kb+ DNA ladder Life Technologies 10787-026
Ethidium bromide Sigma-Aldrich E7637
1.5ml Microcentrifuge tubes VWR (Axygen Scientific) 10011-700
Culture sticks VWR CA10805-018
PCR tubes Diamed AD0210-FCN (new #: DIATEC420-1250)
Centrifuge Eppendorf 5417c
Heat block VWR 13259-030 / 13259-286
Thermalcycler Applied Biosystems (2720) 4359659
Horizontal, submersible gel electrophoresis chamber with gel mold BioRad 170-4469
Power supply BioRad 164-5050
Rocker shaker VWR 40000-300
Molecular Imager Gel Doc System BioRad 1708170

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Cite This Article
McClure-Warnier, J., Conly, J. M., Zhang, K. Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus. J. Vis. Exp. (79), e50779, doi:10.3791/50779 (2013).

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