Study On Rapid Detection Method By Multiple PCR Of Four Species Animal Food Borne Pathogenic Bacteria

Bacterial food poisoning accounted for the top of the total number of China's food poisoning. Animal food is the main reason for food poisoning caused by bacterium. At present, the main method of pathogens detection in food is conventional culture-based microbiological, which needs 3 to 7 days and not easy to operate, and the reagents used are complex ,diverse, and high cost . That's the reason for the methods can not meet the actual needs of commodity circulation. PCR (polymerase chain reaction) technology is specific, sensitive, simple and rapid. Research on detecting pathogenic bacteria in food by PCR technology is ascendent. They are the four major animal food borne pathogenic bacteria including EnterohemorrhagicEscherichia coli O157:H7, Listeria monocytogenes, Yersinia enterocolitica and Pseudomonas fluorescens. A new detection method by multiplex PCR was established in our study, combined with the universal preenrichment technique. The four species pathogenic bacteria which were contaminated in animal food can be detected at the same time by one step in 6 hours after universal preenriching with LB broth medium. The method had such advantages: well specifically, simple, rapid, economical and practical. The results were as follows:(1) The study on the universal preenrichment method of nine species animal food borne pathogenic bacteria was effectuated. They were EnterohemorrhagicEscherichia coli O157:H7, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella typhi, Staphylococcus aureus, Yersinia enterocolitica, Listeria monocytogenes, Shigella sonnei, and Pseudomonas fluorescens. Results showed that the optimum conditions of preenrichment were LB broth medium, cultured at 36℃for 18 h.Under these conditions, the enrichment was the best, balanced and consistent enrichment of magnitude, reaching 108 cfu /mL.)(2) The experiment contains nine species pathogenic bacteria--EnterohemorrhagicEscherichia coli O157:H7, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella typhi, Staphylococcus aureus, Yersinia enterocolitica, Listeria monocytogenes, Shigella sonnei,and Pseudomonas fluorescens. These bacterias were cultured under the conditions of 30℃, 33℃,36℃for 12 h, with LB,NB and UPB medium, the cell population of them can reach 106 cfu/mL. It could meet the needs of detection method, such as PCR. The culture time in 36℃should be controlled around 12 ~ 18 h, not more than 18 h.(3) Four pairs of primer are designed in our study according to the gene: eaeA of EnterohemorrhagicEscherichia coli O157:H7, hly of Listeria monocytogenes, ail of Yersinia enterocolitica, htpX of Pseudomonas fluorescens. The result showed that four pairs of primer had good specificity.(4) The Single PCR and multiplex PCR test system of four species pathogenic bacteria were established in our study and tested in simulation food amples , which were Enterohemorrhagic Escherichia coli O157:H7, Listeria monocytogenes, Yersinia enterocolitica, Pseudomonas fluorescens. In the end, we have groped the optimal parameters of multiplex PCR system and thermal cycling parameters. The methods has good Specificity by a series of experiments.