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Contamination of pathogenic bacteria in food foodborne illness is caused by one of the main factors, rapid detection of pathogenic bacteria in food is a timely and effective prevention of spread of pathogenic bacteria and food poisoning is an important prerequisite. Currently rely mainly on the detection of pathogenic bacteria conventional bacteriological culture method, which normally takes 4 ~ 7d, operating tedious, time-consuming and labor-intensive: Multiplex PCR as a simultaneous detection of a variety of pathogenic bacteria method developed very rapidly. This study established Salmonella, E. coli and Staphylococcus aureus multiplex PCR technique, and initially applied to part of Hubei CIQ export food sample testing, the main findings are as follows: 1 Salmonella, E. coli and Staphylococcus aureus multiplex PCR primer design and specificity analysis (1) under the Salmonella invA gene, E. coli phoA gene and Staphylococcus aureus nuc gene sequence, the application of Primer 5.0 primers were designed by Oligo 6.0 for analysis to ensure that the primer a non-dimer formation, and complementarity between each primer, optimal annealing temperature were analyzed simultaneously using BLAST program, through GeneBank of each primer amplification products and the homology comparison, given the third pair of primers . (2) optimization of Salmonella, Escherichia coli and Staphylococcus aureus PCR reaction conditions, specific primers for the experimental results designed to Salmonella, E. coli and S. aureus-specific primers amplified 284bp, 622bp, 484bp The target band control strains were negative. No cross reaction between the primer and specificity. 2 Salmonella, E. coli and Staphylococcus aureus multiplex PCR assay for detection of optimized multiplex PCR experiments to determine the optimum reaction conditions Salmonella, E. coli and Staphylococcus aureus primer concentrations were 40nmol / L, 40nmol / L, 80nmol / L, Mg 2 concentration of 2.4mmol / L, dNTP concentration of 200μmol / L, Taq DNA polymerase 1.5U. Cycle parameters: 94 ℃ for denaturation 7min; 94 ℃ denaturation 30s, 55 ℃ annealing 30s, 72 ℃ extension 30s, a total of 30 cycles; final extension at 72 ℃ 5min. Under this condition, simultaneous detection of multiple PCR Salmonella, E. coli and Staphylococcus aureus DNA sensitivity are 10.2pg, 10.2pg, 102pg. 3 Meanwhile enrichment of Salmonella, E. coli and Staphylococcus aureus enrichment medium of this study to design a simultaneous enrichment of Salmonella, E. coli and Staphylococcus aureus buffered saline broth (buffed saline broth , BSB), the main component and content: peptone 10g, beef extract 3g, disodium hydrogen phosphate (Na 2 HPO 4 · 12H 2 O) 9g, potassium dihydrogen phosphate 1.5g, additives 50g, distilled water 1000mL, pH7.2. 4 multiplex PCR Application and evaluation copies of artificially inoculated pathogens on 10 milk samples, 20 parts and 50 parts of Hubei sewage samples CIQ export shrimp samples, multiplex PCR and GB methods have nine Salmonella-positive, 27 E. coli were positive, 10 were positive for Staphylococcus aureus, multiplex PCR method was positive and GB have 1 negative Salmonella, three large
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