Optimization and Evaluation of Triplex Real-time PCR Assay for Detection of Genes Encoding Staphylococcal Virulence and Methicillin Resistance Using Two Different Multi-channel Emission Instruments

Abstract

Aim of Study: To optimize a triplex real-time PCR assay developed elsewhere and to evaluate the performance characteristics of two different multi-channel real-time PCR systems on the newly optimized assay. Methodology: A triplex real-time PCR assay developed for three key genes encoding virulence and antibiotic resistance in Staphylococcus aureus, namely, lukSF-PV, mecA, and spa was optimized and evaluated using two different real-time PCR instruments (7500SDS and LightCycler 480). Bacterial strains (N=230), including staphylococcal and non-staphylococcal isolates, were used for the study. Results: Following optimization, cycling and data analysis completed within one hour compared with the former three hours. Assay specificity became 100% on both instruments. The negative predictive value (NPV) and the positive predictive value (PPV) rose to 100%. Conclusion: The optimized triplex real-time PCR assay is highly reproducible across the two systems without loss of speed, sensitivity and specificity. The results also suggested that user-familiarization with each machine operational system would allow assay performance across various real-time PCR platforms currently being used.