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Development and application of a real-time PCR assay for detection of Vibrio parahaemolyticuss in oyster

LIN Qiang1,2,LI Ningqiu1,FU Xiaozhe1,LIU Lihui1,SHI Cunbin1,WU Shuqin1 1.Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China; 2.College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China  
Vibrio parahaemolyticus(VP) is a pathogen that is the leading cause of shellfish-associated cases of bacterial food poisoning.To establish a real-time PCR for quantitative analysis of toxR gene,a pair of specific primers and TaqMan fluorescent probe of toxR gene of VP were designed.The positive recombinant plasmids of toxR gene served as templates to establish standard curve,which showed corresponding relationship between the copies of plasmids and threshold cycle(CT) values.The lowest limit detection was 15 copies of toxR gene for plasmid,and the sensitivity of pure cultures and simulated oyster sample was 18 CFU/mL and 180 CFU/g,respec-tively.The coefficient of variation was 0.95% for intra-assay test and 1.5% for inter-assay test.One hundred and sixty eight positively established samples were detected from 178 oysters collected from a farm in Guangdong Province.The result showed that the real-time PCR assay for VP had high specificity,sensitivity,repeatability and it was time saving,so it can be used for detecting and monitoring VP in aquatic products.
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