Abstract

In this study, a novel multiplex- polymerase chain reaction (PCR) for rapid detection of various staphylococci strains, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus, (MSSA) methicillin-resistant coagulase-negative staphylococci (MRCNS), methicillin-sensitive coagulase-negative staphylococci (MSCNS) and non-staphylococci strains, had been developed and applied. Six primers were specially designed on three target genes, which were mecA, 16S Ribosomal ribonucleic acid (rRNA)and femA. The specific amplification generated 3 bands on agarose gel, with sizes 374 bp formecA, 542 bp for 16S rRNA and 823 bp for femA, respectively. The PCR product showed highest levels of resolution of DNA when 250 μM of dNTP, primer concentration of mecA, 16S rRNA and femA reaching 1, 1 and 3 μM respectively. No false positive amplification was observed, indicating the high specificity of the established multiplex PCR assay. Application of this multiplex-PCR had been further performed on detection for 262 MRSA and MRCNS strains with primers pairs M1 with M2 and F1 and F2. According to the results, multiplex-PCR results showed expected products for either MRSA or MRCNS strains, demonstrating the multiplex-PCR assays established in this study to be useful and powerful methods for differentiation of MRSA, MSSA, MRCNS, MSCNS and non-staphylococci strains.

Key words: Staphylococcus, multiplex-PCR, rapid detection.