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《Animal Husbandry & Veterinary Medicine》 2019-01
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Establishment and application of a triple Real-time PCR for detection of Vibrio parahaemolyticus

WAN Ying;LING Jiao;WANG Quan;SUN Weidong;JIANG Wei;College of Veterinary Medicine,Nanjing Agricultural University;Shanghai Veterinary Research Institute,CAAS;  
To establish a rapid,accurate and efficient method for detection of Vibrio parahaemolyticus (VP),three pairs of specific primers and corresponding Taq Man probes were designed according to the species-specific gene toxR and the virulence genes tdh and trh of V. parahaemolyticus. The 5-ends of the probes for toxR,tdh and trh were individually labeled with FAM,HEX and CY5. The 3' -ends were all labeled with quencher BHQ1. The concentration and reaction parameters of each reaction were optimized to build a triple Real-time PCR based on the Taq Man probe method for the quantitative detection of V. parahaemolyticus. The results showed that the specificity of the assay had no cross-reaction with other pathogens,the minimum detection limit was 10 CFU/m L,and the variation coefficient of the repeated experiments was less than 1%. The triple Real-time PCR was used to detect shrimp and shellfish samples contaminated by V. parahaemolyticus,and the minimum detection reached 10 CFU/m L. The whole detection lasted approximately 1 h. A conclusion could be drawn that a new triple Realtime PCR method established in this study possessed strong specificity,high sensitivity,good repeatability and ideal convenience. It was an effective method for high-throughput detection of pathogenic V. parahaemolyticus.
【Fund】: 上海市科学技术委员会科研计划项目(17140900400);; 国家重点研发计划项目(2017YFC1200201)
【CateGory Index】: S941.4
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