Study On Detection Kit Of Food-borne Pathogens By The Multiplex PCR

The problem of food safety is becoming more awared and more serious in many countries .It's associated closely with the national development and people's health and happiness. The immediate and proper treatment to patients who have eaten contaminated food is important as well as the food safty testing before food are sold or eaten by people. What's urgent now is how to detect pathogenic bacteria in food and patients. To develop an effective and practical way to detect foodborn pathogens in this study, six species of foodborne pathogens are detected specifically and successfully in multiplex PCR system by amplifying each target gene segment.In this study, took use of Staphylococcus aureus of special sequences of conservative nuc gene, Salmonella spp inv gene, Shigella spp ipaH gene, listeria monocytogenes hlyA gene, Bacillus cereus hblA gene, E.coli-0157: H7 hlyAB gene according to the result of BLAST to design six special primers. The two triplex PCR methods were developed for the simultaneous detection of the six pathogens from meat. In the PCR process, DNA concentration, Mg2+ concentration, template volume, annealing temperature and PCR circles are optimized to determine the optimal PCR. The reaction mixture consisted of 50uL: 5 uL10×PCR buffer, 75pmol Mg2+ , 10pmol mixture of dNTPs,,5pmol hlyAB,5pmol ipaH和20pmol hblA; 7.5 pmol invA,10 pmol hlyA and 10pmol nuc,5U Taq enzyme. The reaction was run under the following conditions: Cool start; DNA pre-denaturation at 94℃for 5 min, DNA denaturation at 94℃for 30s, primer annealing at 58℃for 40 s ,and DNA extention at 72℃for 70s, run 28 cycles; the final extention was performed at 72℃for 12 min. PCR products were confirmed by DNA sequencing. The results of sequencing compared with the target gene sequence at gene bank showed that PCR amplified products were certified.The sensitivity of the simultaneous detection of the six bacterial pathogens with m-PCR when using purified DNA of the bacterial type strains was 4.5 cfu/mL for Staphylococcus aureus, 4.7 cfu/mL for Salmonella spp, 17 cfu/mL cfu/ml for Shigella spp, and 17 cfu/mL for listeria monocytogenes, 19.8 cfu/mL for E.coli-0157: H7, for 17.7cfu/mL Bacillus cereus.In our study, we developed the two triplex PCR methods for simultaneous detection of six common food-borne bacterial pathogens in various samples collected from chicken abattoirs, processing plants and retail vendors in Tai'an, Shandong Province of China. A total of 502 samples including chicken feces and feathers, chicken washes, chicken carcasses from processing plants, fresh raw chickens, and frozen chickens were collected and used for the validation of the triplex PCR method. Salmonella was identified in 31.7% of the samples, whereas Bacillus cereus, E.coli-0157: H7, L. monocytogenes, Shigella and Sta. aureus were detected in 11.2%, 9.0%, 5.2%, 0.6% and 0.6% respectively. In this study, real detection was made. The result indicates: the sensitivity of the m-PCR assay is 100%.Conclusion: The detection kit has the superiority in theory and practical application, and also examinessix pathogens once. It can be used in clinical diagnosis.These results also demonstrate that the six common food-borne bacterial pathogens are prevalent in raw poultry products in Tai'an, China, and indicate the necessity to screen bacterial pathogens routinely during the poultry processing procedures using quick detection methods to reduce the risk of food-borne diseases.