Evaluation of three different molecular markers for the detection of Staphylococcus aureus by polymerase chain...

The aim of this study was to target three genes of Staphylococcus aureus-fmhA (coding for a factor of unknown function), catalase and femA (coding for a factor essential for methicillin resistance) to establish and validate a PCR assay for the detection of this pathogen. Two pairs of primers were designed for fmhA and one pair each for catalase and femA genes. The PCR assays were standardized and found to give specific amplicons under similar reaction parameters. Target specificity of the primers was confirmed by DNA sequencing of the amplicons. While the initial inclusivity and exclusivity test reactions were in agreement in case of three of the primer pairs, one pair based on fmhA gene produced a non-specific product with a template DNA used in exclusivity test reactions. Forty-five strains of S. aureus were subjected to these PCR assays for their evaluation. Three among the four pairs of primers, one against each gene detected all the 45 strains precisely whereas one of the PCR assays using primers targeting the fmhA gene did not generate the specific amplicon with several of the strains. Seven unidentified strains of Gram-positive cocci subjected to these PCR assays produced negative results for each culture. Six of the strains were identified as Staphylococcus haemolyticus and one strain as Staphylococcus arlettae by 16S ribosomal gene analyses. All the three assay systems showed a detection limit of 100 cells per 20mul reaction assay. For validation of these assay systems, 80 coded samples of 11% skimmed milk spiked with different pathogens were received from NICED (National Institute of Cholera and Enteric Diseases), Kolkata and subjected to these PCR assays. All the three assays could detect S. aureus correctly in two of the samples. Amongst 150 raw milk samples, 36 (24%) were found positive for S. aureus. We conclude that fmhA, catalase and femA genes are conserved in S. aureus and, therefore, could be used as specific targets for its detection and identification by PCR. The protocols developed herein could be used for rapid and specific detection of this pathogen in food, clinical and environmental samples, especially milk.