双通道荧光PCR快速检测水产品中主要致病性弧菌的研究  

Study on the Duplex Real-time PCR Assay for Rapid Detection of Main Pathogenic Vibrio in Aquatic Products

作　　者：帅江冰 傅玲琳 张晓峰 励建荣 

机构地区：[1]浙江出入境检验检疫局,杭州310012 [2]浙江工商大学食品与生物工程学院浙江省食品安全重点实验室,杭州310035

出　　处：《中国食品学报》2011年第5期151-157,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：浙江出入境检验检疫局科技项目(ZK200951);"十一五"国家863计划海洋技术领域重点项目(2007AA091806)

摘　　要：目的:建立水产品中副溶血弧菌和霍乱弧菌快速、敏感、特异的双通道荧光PCR同步鉴别体系。方法:针对副溶血弧菌TDH基因和霍乱弧菌ctxA基因设计合成2对特异性引物和2条Taqman探针,优化体系,建立双通道荧光PCR体系。结果:建立的双通道荧光PCR体系特异性强,引物和探针之间无干扰。对副溶血弧菌和霍乱弧菌的检测灵敏度高,下限均能达到5×104CFU/mL。体系稳定,重复性好,可操作性强,两种不同浓度基因组DNA无相互抑制现象,适用于不同条件、多种样品的检测。采用该方法对140份采自舟山、象山和杭州的各类水产品进行检测,副溶血弧菌和霍乱弧菌检出率分别为25.7%和3.6%,未见霍乱弧菌和副溶血弧菌混合污染的样品,表明建立的双重荧光PCR是一种可用于水产品等样品中副溶血弧菌和霍乱弧菌的快速、灵敏、特异的鉴别方法。Objective：To develope a duplex real-time PCR assay for rapid detection of Vibrio cholerae and Vibrio parahaemolyticus simultaneously in aquatic products.Methods：two primer pairs and corresponding probes were designed against TDH gene of Vibrio parahaemolyticus and ctxA gene of Vibrio cholerae,respectively,to develop the duplex real-time assay.Results：the two pairs of primers and probes used in the assay were specific and had no cross-reations in detection of two genomic DNA.The duplex real-time PCR assay had a higher sensitivity than conventional PCR method,with detection limits of 5×104 CFU/mL for each bacterial.The results also showed that the assay was reproducible,and diplayed no inhibition to efficiency of two separated reactions in one tubes with different concentrations of two genomic DNA,indicating that the assay was suitable for clinical diagnosis.The duplex real-time PCR assay was then successfully applied to detection of Vibrio cholerae and Vibrio parahaemolyticus in samples of 140 seafoods collected from Hangzhou,Xiangshan and Zhoushan,China,which showed the positive rates of 3.6% and 25.7,respectively.However,there was no coinfecion of both bacterias detected.All the results indicating that the duplex real-time PCR assay was a rapid,sensitive,efficient and feasible method.

关 键 词：副溶血弧菌 霍乱弧菌 双通道荧光PCR TAQMAN探针 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]