In silico PCR Verification and Simplex Real-Time PCR Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) from East Coast Malaysian Clinical Isolates

Suhaili, Zarizal and Ali, Abd Manaf and Sajili, Mohammad Hailmi and Yahya, Azifah (2013) In silico PCR Verification and Simplex Real-Time PCR Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) from East Coast Malaysian Clinical Isolates. Walailak Journal of Science and Technology (WJST), 10 (3). pp. 237-246. ISSN 16863933

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Abstract

The aims of this study were to validate the primers developed for molecular-based detection and identification of Malaysian clinical isolates of methicilin-resistance Staphylococcus aureus (MRSA) using in-silico Polymerase Chain Reaction (PCR) and real-time PCR SYBR with Green I. Rapid molecular diagnostic and risk assessment of the MRSA are possible by real-time PCR SYBR Green I. However, validation of such primers for real-life samples is expensive and time consuming. Hence, development and verification of real-time PCR primers by in-silico PCR can be the first step in the selection of the most appropriate primers. Three species-specific markers were chosen targeting coa (staphylocoagulase), nuc (thermonuclease) and mecA (methicillin-resistance) and were specifically verified against 35 selected S. aureus strains by using in-silico PCR. For the actual laboratory verification, all of the 3 genes were detected with a single specific melting curve peak (Tm at 76.16 ± 0.8 °C, 78.50 ± 0.4 °C and 74.41 ± 0.6 °C for a coa, nuc and mecA respectively) in 32 bacterial strains including ATCC reference strains. Thus, there is no disagreement between both in-silico PCR and real-time PCR verification and validation of the primers designed for the detection and identification of MRSA in this study. The potential of using a bioinformatics approach (in-silico PCR) before selecting primer pairs for a given study may enable researchers to accept or reject the potential primer pairs for downstream experimental (in vitro) PCR without wasting any chemicals as well as related cost.

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