Multiplex PCR Ensaio para a digita
1Centre for Antimicrobial Resistance,Alberta Health Services / Calgary Laboratory Services / University of Calgary, 2Department of Pathology & Laboratory Medicine,University of Calgary, 3Department of Microbiology, Immunology and Infectious Diseases,University of Calgary, 4Department of Medicine,University of Calgary, 5The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases,University of Calgary
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
Estafiloc
Introduction
Staphylococcus aureus resistente
Protocol
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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…
Divulgazioni
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 |
Riferimenti
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Citazione di questo articolo
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).