The Establishment And Application Of Nested PCR For Rapid Detection Of Vibrio Parahaemolyticus In Seafood

Vibrio parahaemolyticus is one of main pathogenic bacteria that causes foodborne disease. Epidemic diseases caused by V. parahaemolyticus is ascending year by year. In this study, we mainly investigated contamination status and the cases of virulence genes carrying for V. parahaemolyticus from aquatic products in Jinzhou Bijia Mountain coasts and established a nested-PCR detection assay for V. parahaemolyticus. This method offers references to detection of V. parahaemolyticus. At the same time, we compared nested-PCR with national standard of GB/T 4789.7-2008. The results demonstated that they showed the same outcomes, but nested-PCR had more advantages than the traditional method in the detection time.The main conclusions of this paper are as follows:1. Three hundred and nineteen samples of different varieties of aquatic products were randomly sampled from the coast of Jinzhou Bijia Mountain. The total detection rate of V. parahaemolyticus reached 29% according to the method of GB/T 4789.7-2008 and 16 S rRNA, which demonstrated that the aquatic products from Bijia Mountain coasts were seriously polluted by V. parahaemolyticus. Aquaculture farmers, professionals and customers should pay attention to the pollution of aquatic products by V. parahaemolyticus. The necessary measures should be taken to prevent outbreaks of foodborne diseases.2. One hundred and twenty four strains of V. parahaemolyticus isolated from samples was tested by virulence related gene. The results show 2 strains which carring the gene of tdh were found, suggested the detection rate was 1.6%. Strains with the gene of tdh show the beta hemolytic phenomena in kanagawa hemolysis test. Two strains which carring the gene of trh was found, suggested the detection rate was 1.6%. Strains with the gene of trh show positive urease in the test of urea enzyme.3. The reaction system and conditions for nested-PCR were established by optimizing the usages of DNA, primers, Mg2+, dNTPs, rTaq DNA and the annealing temperature. This method can be finished in 4h, saving a lot of time for testing. The reaction system used the gene of Vibrio alginnolyficus, Vibrio vulnificus, Escherichia coil, Bacillus licheniformis, Rhamnose salmonella, Staphylococcusaureus as a template for amplification. The results showed that no purpose stripe were founded, which illustrated the nested-PCR had good specificity. Sensitivity test results showed that the detection limit were 6.62cfu/mL for the pure culture of V.parahaemolyticus and 200 fg for DNA. The artificial pollution tests showed that V. parahaemolyticus can be checked out after artificial propagation of bacteria for 2h(the initial concentration was 2.57cfu/mL), proved that nested-PCR had moderate sensitivity. We established a new method of nested-PCR to test V. parahaemolyticus in aquatic products, owing to the results between nested-PCR and GB/T 4789.7-2008 were same.