Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

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

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) has acquired and integrated into its genome a 21-67-kb mobile genetic element, termed the staphylococcal cassette chromosome mec (SCCmec) that harbors the methicillin resistance (mecA) gene and other antibiotic resistance determinants ¹. SCCmec is characterized by the presence of terminal direct and inverted repeats, two essential genetic components (the mec gene complex and the ccr gene complex), and the J-regions (Figure 1). The mec gene complex is composed of IS431mec, mecA, and intact or truncated sets of regulator....

Protocol

These procedures should be conducted in a certified biosafety level 2 laboratory. Appropriate personal protective equipment such as gloves and lab coats should be used at all times.

1. Sample Preparation

1. Fresh overnight plate cultures of MRSA are required. On the day before PCR is to be done, select a single colony of MRSA with a sterile culture stick and prepare a heavy streak of the bacteria on a Tryptic Soy Agar (TSA) plate. Multiple samples can be streaked onto a single plate. Incubate overnight at 37 °C.
2. Add 75 μl of sterile distilled water to a 1.5 ml microcentrifuge tube. Using a sterile culture stick ....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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