Abstract
BackgroundStaphylococcus 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.ResultsThe 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.ConclusionThe 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.
Highlights
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
The analytical specificity of the pentaplex PCR assay at the genus level was determined using 10 staphylococcal reference strains and found to be positive for the Staphylococcus genus specific 16S rRNA gene
All the reference strains of S. aureus were positive for femA gene by pentaplex PCR, while other Coagulase-negative staphylococci (CoNS) species were negative (Table 1)
Summary
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. An increasing number of MRSA cases has been encountered globally among healthy community residents [3]. These isolates are referred to as community-acquired MRSA (CA-MRSA), which are genetically and phenotypically different from representative hospitalacquired MRSA (HA-MRSA), in relation to their antibiotic resistance patterns, and by the allocation of their staphylococcal chromosomal cassette (SCCmec) types, IV and V [3,4]. The mecA gene, the structural determinant that encodes PBP2a, is considered as a useful molecular marker of putative methicillin resistance in S. aureus and CoNS [9,10]
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