水产品中霍乱弧菌、副溶血弧菌和创伤弧菌多重PCR检测方法的建立

DEVELOPMENT OF A MULTIPLEX POLYMERASE CHAIN REACTION ASSAY FOR SIMULTANEOUS DETECTION OF VIBRIO PARAHAEMOLYTICUS, VIBRIO CHOLERAE AND VIBRIO VULNIFICUS IN AQUATIC FOODS

霍乱弧菌、副溶血弧菌和创伤弧菌是引起全球范围的食源性疾病的重要病原菌,建立一种能准确鉴别这3种弧菌的方法具有重要意义。groEL基因是包括弧菌属在内的多种细菌检测的分子标记。本研究根据3种弧菌的groEL基因分别设计引物,经PCR扩增及反应条件的优化,建立一种能同时检测这3种弧菌的多重PCR方法。结果表明应用多重PCR技术对霍乱弧菌、副溶血弧菌和创伤弧菌能分别特异性地扩增出418、644、192 bp的目的片段。检测其他非目标供试菌时,不出现任何扩增条带。敏感性检测结果表明,对霍乱弧菌、副溶血弧菌和创伤弧菌最低检测限分别为102、102、103CFU/m L。人工染菌水产品检测结果显示,所建立的多重PCR方法能够有效检测出目的细菌,而未染菌组均为阴性。本文通过建立的多重PCR方法实现了对这3种弧菌的同时检测,具有快速、灵敏度高和特异性强等优点,对水产品中这3种弧菌的检测和流行病学调查具有重要意义。

Vibrio cholerae（Vc）, Vibrio parahaemolyticus（Vp） and Vibrio vulnifi cus（Vv） are of major concerns in aquatic food industry as they are important pathogens causing food-borne diseases throughout the world. Therefore, it is very necessary to develop an effective multiplex polymerase chain reaction（PCR） for the simultaneous detection of these important Vibrio species. The gro EL gene is a potential marker for simultaneous detection of these species in seafood samples. Three sets of species-specifi c primers were designed to develop a multiplex PCR. The reaction conditions of the multiplex PCR were optimized to establish a rapid, sensitive and convenient PCR method. The results showed that the multiplex PCR produced amplicons with expected sizes of 418 bp, 644 bp and 192 bp specifi c for Vc, Vp and Vv, respectively. This PCR method showed good effi ciency as it detected three species in the same samples. The detection limits were demonstrated to be 102CFU/m L for Vp and Vc and 103CFU/m L for Vv in the mixed DNA samples. In addition, the multiplex PCR specifically amplified target species but did not cross-react with other Vibrio and non-Vibrio species. The results indicated that the multiplex PCR was a quick and cost-effi cient method for detection of Vibrio species in seafoods and for epidemiological investigation.