Study On The Detection Of Salmonella Spp., Escherichia Coli And Staphylococcus Aureus By Multiplex PCR

Food contamination with bacterial pathogens are major reasons of food-borne disease. Rapid detection of food-borne bacteria is essential to efficiently prevent pathogens prevalence and food poisoning. Conventional methods of detecting bacteria in food relies on selective medium, microbiological and biochemical test can often take more than 4～7 days to complete, which are cumbersome and time-consuming. Multiplex PCR which can simultaneously detect more than one bacterium has been applied increasingly comprehensive. In this study, multiplex PCR assays were developed for the simultaneous detection of Salmonella spp., Escherichia coli and Staphylococcus aureus and used in some food samples from HuBei entry-exit inspection and quarantine bureau. The results are as follows:1. Results of primer design for multiplex PCR and specificity test(1) According to the invA gene of Salmonella spp., the phoA gene of Escherichia coli and the nuc gene of Staphylococcus aureus, three pairs of primers were designed by Primer 5.0 and were analyzed by Oligo 6.0 to insure the close anneal temperature and avoid the formation of steady dimer. Moreover the primers and amplification fragments were analyzed by BLAST to avoid high homology and complementarity through GeneBank. (2)The PCR amplification conditions were optimized and the primers specificity test were developed. The results showed that the three pairs of primers produced specific amplicons of expected sizes, 284bp for Salmonella spp., 622bp for Escherichia coli, 484bp for Staphylococcus aureus.2. Development of multiplex PCR assay for Salmonella spp., Escherichia coli and Staphylococcus aureusThe optimized reaction conditions followed as the concentration of primer 40nmol/L for Salmonella spp., 40nmol/L for Escherichia coli, 80nmol/L for Staphylococcus aureus, 2.4mmol/L Mg²⁺, 200 μ mol/L dNTP, 1.5U Taq DNA polymerase. The reaction was run under the following conditions: DNA pre-denaturation at 94 °C for 7min, DNA denaturation at 94 °C for 30s, primer annealing at 55 °C for 30s, and DNA extension at 72 °C for 30s for 30 cycles, the last extension was performed at 72°C for 5min. Under the...