Development Of Multiplex PCR And Gene Chip Mehtods For The Detection Of Staphylococcus Aureus

A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. 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 56oC. This multiplex PCR method was performed with 71 strains of Staphylococcus aureus and 51 strains of 6 other bacterial species. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and the primers for coa were specific for S. aureus. Among the S. aureus strains tested, 39.4% (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 2.4×10~3 CFU of S. aureus.In addition, the gene chip technology was developed based on the multiplex PCR method to detect another three entertoxin genes (sed, seg and sei genes) and one S. aureus specific control gene femB. The adjustments of the annealing temperature of multiplex PCR and gene chip were carried out. The sensitivity of the gene chip method was 0.3 ng per reaction?which equals to 108 copies of S. aureus genome.