DEVELOPMENT OF A MULTIPLEX PCR METHOD FOR DETECTION OF THE GENES ENCODING 16S rRNA, COAGULASE, METHICILLIN RESISTANCE AND ENTEROTOXINS IN STAPHYLOCOCCUS AUREUS

Abstract

ABSTRACT A multiplex polymerase chain reaction (PCR) method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), SEs A, B and C (sea, seb and sec), coagulase (coa), and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and the primers for coa were specific for Staphylococcus aureus. Based on the results, the multiplex PCR was accomplished at an optimal Mg2+concentration of 1.0 mM and at an annealing temperature of 56C. This multiplex PCR method was performed with 71 strains of S. aureus and 51 strains of six other bacterial species. Among the S. aureus strains tested, 40.0% (28/71) were found to contain the mecA gene. One of the 28 mecA+strains was not resistant to methicillin. The sea, seb and sec genes were present in 47.9% (34/71), 5.6% (4/71) and 8.5% (6/71) of S. aureus strains, respectively. The sensitivity of this multiplex PCR method was approximately 104.5 pg of genomic DNA per reaction, which was equivalent to an estimated 2.4 × 103 cfu of S. aureus or 3.64 × 104 copies of genome equivalent.PRACTICAL APPLICATIONSThe developed multiplex polymerase chain reaction (PCR) method will be a useful tool for the detection and identification of Staphylococcus aureus from foods, clinical samples and environmental surveys. In particular, the routine use of this multiplex PCR method for the detection of foodborne S. aureus could be used to monitor the presence of enterotoxins and the emergence of methicillin resistance in a population that, to date, has had a relatively low incidence of methicillin resistance.