Multiplex PCR-test voor het typen van Staphylococcus Cassette Chromosome Mec Types I tot en met V in Methicilline-resistente<em> Staphylococcus aureus</em
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
Staphylococcen Cassette Chromosome mec (SCC mec) typen is een zeer belangrijke moleculaire tool voor het begrijpen van de epidemiologie en klonale stam verwantschap van methicilline-resistente Staphylococcus aureus (MRSA), met name de opkomende uitbraken van de gemeenschap-geassocieerde MRSA (CA-MRSA) die zich op een wereldwijde basis. Traditionele PCR typering's classificeren SCC mec door targeting en identificeerbaar is mec en ccr gen complexe vormen, maar vereisen het gebruik van veel primer sets en meerdere afzonderlijke PCR experimenten. We ontwierpen en publiceerde een eenvoudige multiplex PCR-test voor het snel-screening van grote SCC mec types en subtypes I tot V, en later bijgewerkt als beschikbaar werd nieuwe sequentie-informatie. Deze eenvoudige test richt zich op individuele SCC mec types in een enkele reactie, is het gemakkelijk te interpreteren en is uitgebreid wereldwijd gebruikt. Vanwege de verfijnde nature van de test en het grote aantal primers in de reactie, is de kans op problemen tijdens de aanpassing van deze assay aan elk laboratorium. Om het proces van de oprichting van een MRSA SCC mec test te vergemakkelijken, hier laten we zien hoe het opzetten van onze multiplex PCR-test, en bespreken een aantal van de essenti
Introduction
Methicilline-resistente Staphylococcus aureus (MRSA) is overgenomen en ge
Protocol
Representative Results
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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