Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/10/2008
Publication Date: 12/1/2008
Citation: Fan, Y., Pan, F., Paoli, G., Xiao, Y., Sheng, H., Shi, X. 2008. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus. Journal of Rapid Methods and Automation in Microbiology.16(4):394-411. Interpretive Summary: Staphylococcus aureus is an important disease causing bacterium sometimes present in food. The capacity of Staphylococcus aureus to cause disease, in particular food poisoning, is due to the bacterium’s ability to produce a variety of toxins (staphylococcal enterotoxins). Recently, methicillin-resistant Staphylococcus aureus (MRSA) has become a concern due to an increase in the number of hospital-acquired MRSA infections. This study describes the development of a DNA-based method (multiplex PCR) that allowed the detection S. aureus and determined the bacterium’s potential for the production of enterotoxins and methicillin resistance. This single-tube assay was used to characterize a large collection of Staphylococcus and other bacteria. When coupled with microbiological techniques for isolation of Staphylococcus, this multiplex PCR method could be used to detect and characterize Staphyloccocus aureus from foods, clinical, or environmental samples. 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.
Technical Abstract: A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins 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 S. aureus. Amplification conditions, including Mg2+ concentration and annealing temperature, were evaluated. Based on the results, the multiplex PCR was accomplished at an optimal Mg2+ concentration of 1.0 mM and an annealing temperature of 56 deg C. This multiplex PCR method was performed with 71 strains of Staphylococcus aureus and 51 strains of 6 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 46.6% (34/73), 5.5% (4/73) and 8.2% (6/73) 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.