A pentaplex PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus and Panton-Valentine Leucocidin

Abstract

Staphylococcus aureus is a major human pathogen, especially methicillin-resistant S. aureus (MRSA), which causes a wide range of hospital and community-acquired infections worldwide. Conventional testing for detection of MRSA takes 2–5 days to yield complete information of the organism and its antibiotic sensitivity pattern. The present study focused on the development of a pentaplex PCR assay for the rapid detection of MRSA. The assay simultaneously detected five genes, namely 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, lukS that encodes production of Panton-Valentine leukocidin (PVL), a necrotizing cytotoxin, and one internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. The analytical sensitivity and specificity of the pentaplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the pentaplex PCR at the DNA level was found to be 10 ng DNA. The analytical specificity was evaluated with 34 reference staphylococci and non-staphylococcal strains and was found to be 100%. The diagnostic evaluation of MRSA carried out using 230 clinical isolates, showed 97.6% of sensitivity, 99.3% of specificity, 98.8% of positive predictive value and 98.6% of negative predictive value compared to the conventional method. The presence of an internal control in the pentaplex PCR assay is important to exclude false-negative cases. The pentaplex PCR assay developed was rapid and gave results within 4 h, which is essential for the identification of Staphylococcus spp., virulence and their resistance to methicillin. Our PCR assay may be used as an effective surveillance tool to survey the prevalence of MRSA and PVL-producing strains in hospitals and the community.