摘要
目的建立一种同时检测副溶血性弧菌两种毒力基因tdh和trh的双重荧光聚合酶链式反应(PCR)方法,并对我国2771株食源性副溶血性弧菌携带的毒力基因进行全面检测。方法针对副溶血性弧菌tdh和trh毒力基因分别设计荧光PCR引物和探针,优化荧光PCR反应体系及反应程序,建立可同时检测两种毒力基因的双重荧光PCR检测方法。应用所建方法对我国2015年和2016年分离的2771株食源性副溶血性弧菌携带的毒力基因进行检测,并与PCR方法检测结果进行比对,评价方法的灵敏性、准确性和特异性。结果建立的双重荧光PCR方法可同时检测tdh和trh两种毒力基因,其灵敏度达1.5×10^(-4)ng/μL,准确性和特异性均为100%。我国2015年食源性副溶血性弧菌中tdh和trh基因携带率分别为(0.26%,3/1137;1.67%,19/1137),2016年食源性副溶血性弧菌中tdh和trh基因携带率分别为(0.24%,4/1634;0.43%,7/1634)。结论本研究建立了一种同时检测副溶血性弧菌tdh和trh毒力基因的双重荧光定量PCR方法,能够快速、准确地筛查副溶血性弧菌毒力基因;我国食品来源副溶血性弧菌分离株tdh和trh毒力基因携带率较低;双重荧光PCR方法可应用于食品中副溶血性弧菌致病性研究,为我国居民膳食暴露副溶血性弧菌的风险评估工作提供科学数据。
Objective A dual real-time polymerase chain reaction(PCR)method for simultaneous detection of two virulence genes tdh and trh of Vibrio parahaemolyticus was established,and the virulence genes carried by 2771 strains of foodborne Vibrio parahaemolyticus in China were comprehensively detected.Methods According to the tdh and trh virulence genes of Vibrio parahaemolyticus,PCR primers and fluorescent probes were designed respectively,the real-time PCR reaction system and reaction procedure were optimized,and a dual real-time PCR detection method which can detect the two virulence genes at the same time was established.The virulence genes carried by 2771 strains of foodborne Vibrio parahaemolyticus isolated in 2015 and 2016 were detected by the established method,and compared with the result of PCR method to evaluate the sensitivity,accuracy and specificity of the method.Results The established dual fluorescence PCR method could detect both tdh and trh virulence genes at the same time,and its sensitivity was 1.5×10^(-4)ng/μL.The accuracy and specificity were 100%.In 2015,the carrying rates of tdh and trh genes in foodborne Vibrio parahaemolyticus in China were 0.26%(3/1137)and 1.67%(19/1137)respectively,and were 0.24%(4/1634)and 0.43%(7/1634)in 2016 respectively.Conclusion In this study,a dual real-time PCR method for simultaneous detection of tdh and trh virulence genes of Vibrio parahaemolyticus was established,which could quickly and accurately screen the virulence genes of Vibrio parahaemolyticus.The carrying rate of tdh and trh virulence genes of Vibrio parahaemolyticus isolated from food in China was low.The dual real-time PCR method can be applied to the study of the pathogenicity of Vibrio parahaemolyticus in food,and provide scientific data for the risk assessment of dietary exposure to Vibrio parahaemolyticus in China.
作者
白瑶
李斌
李凤琴
杨大进
徐进
董银苹
王伟
闫琳
江涛
BAI Yao;LI Bin;LI Fengqin;YANG Dajin;XU Jin;DONG Yinping;WANG Wei;YAN Lin;JIANG Tao(Key Laboractory of Food Safety Assessment of Ministry of Health,China National Center for Food Safety Assessment,Beijing 100021,China;China National Research Institute of Food and Fermentation Industries Co.,Ltd,Beijing 100026,China)
出处
《中国食品卫生杂志》
CSCD
北大核心
2022年第1期54-59,共6页
Chinese Journal of Food Hygiene
基金
国家重点研发计划(2019YFC1606404-2)。
