多重PCR检测四种食源性病原弧菌  

Simultaneous Detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae Using Multiplex PCR

作　　者：魏霜[1] 冼钰茵[1] 赵晖 吴希阳[1,3] 

机构地区：[1]暨南大学理工学院食品科学与工程系,广州510632 [2]湖北省出入境检验检疫局检验检疫技术中心,武汉430022 [3]广东省食品安全卫生应急技术研究中心,广州510632

出　　处：《中国农业科学》2013年第8期1682-1686,共5页Scientia Agricultura Sinica

基　　金：公益性行业科研专项项目(201110039)

摘　　要：【目的】建立在扩增内标存在下同时快速检测溶藻弧菌、副溶血弧菌、创伤弧菌、霍乱弧菌4种食源性致病菌的五重PCR方法。【方法】以细菌16S rRNA基因为扩增内标靶序列,针对溶藻弧菌gyrB基因、副溶血弧菌collagenase基因、创伤弧菌vvhA基因、霍乱弧菌ompW基因,分别设计特异性引物,建立多重PCR体系,对其灵敏度和特异性进行评价,并将建立的五重PCR应用于弧菌的筛选。【结果】建立的多重PCR检测体系对混合模板中副溶血弧菌、溶藻弧菌、创伤弧菌的灵敏度达10 CFU/mL,霍乱弧菌的灵敏度达105CFU/mL,经特异性评价证实其特异性好,并能有效指示PCR反应的假阴性,将建立的多重PCR应用于69株疑似弧菌菌株的鉴定,其结果与生理生化鉴定结果一致。【结论】该方法能够快速、灵敏、准确地检测溶藻弧菌、副溶血弧菌、创伤弧菌和霍乱弧菌4种食源性致病菌,灵敏度高,并能有效指示PCR反应的假阴性,适用于食品中常见致病性弧菌的快速筛检。[ Objective ] The objective of this study is to develop a multiplex PCR assay that can simultaneously detect Fibrio alginolyticus, Fibrio parahaemolyticus, Fibrio vulnificus and Fibrio cholerae in the presence of an internal amplification control （IAC）. [Method] Species-specific PCR primers were designed based on gyrB gene for Vibrio alginolyticus, collagenase gene for I/ibrio parahaemolyticus, whA gene for Fibrio vulnificus and ompW gene for Fibrio cholerae, 16S rRNA gene of bacteria as IAC primers was used to indicate false-negative results. Multiple PCR method was developed after optimization reaction condition. [ Result] The multiple PCR method was proved to be rapid, high-throughput, sensitive and specific and the existence of IAC could successfully eliminate false-negative results. The multiplex PCR was validated with 69 suspicious Fibrio strains and the results were consistent with the physiological and biochemical experiments.[ Conclusion ]The multiple PCR method is specific, stable and reliable for the detection of Fibrio alginolyticus, Fibrio parahaemolyticus, Fibrio vulnificus and Fibrio cholerae.

关 键 词：弧菌 检测 多重PCR 

分 类 号：TS207.4[轻工技术与工程—食品科学]