双重实时荧光PCR检测副溶血性弧菌毒力基因方法的建立和应用
作者:
作者单位:

1.国家食品安全风险评估中心,国家卫生健康委员会食品安全风险评估重点实验室,北京 100021;2.中国食品发酵工业研究院有限公司,北京 100026

作者简介:

白瑶 女 副研究员 研究方向为食品微生物 E-mail:baiyao@cfsa.net.cn

通讯作者:

江涛 男 研究员 研究方向为食品微生物 E-mail:jiangtao001@cfsa.net.cn

中图分类号:

R155

基金项目:

国家重点研发计划(2019YFC1606404-2)


Establishment and application of dual real-time PCR for detection of virulence genes of Vibrio parahaemolyticus
Author:
Affiliation:

1.Key Laboractory of Food Safety Assessment of Ministry of Health, China National Center for Food Safety Assessment, Beijing 100021, China;2.China National Research Institute of Food and Fermentation Industries Co., Ltd, Beijing 100026, China

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    摘要:

    目的 建立一种同时检测副溶血性弧菌两种毒力基因tdhtrh的双重荧光聚合酶链式反应(PCR)方法,并对我国2 771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tdhtrh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2 771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tdhtrh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tdhtrh基因携带率分别为(0.26%,3/1 137;1.67%,19/1 137),2016年食源性副溶血性弧菌中tdhtrh基因携带率分别为(0.24%,4/1 634;0.43%,7/1 634)。 结论 本研究建立了一种同时检测副溶血性弧菌tdhtrh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tdhtrh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。

    Abstract:

    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 2 771 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 2 771 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% (3/1 137) and 1.67%(19/1 137) respectively, and were 0.24%(4/1 634) and 0.43%(7/1 634) 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.

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白瑶,李斌,李凤琴,杨大进,徐进,董银苹,王伟,闫琳,江涛.双重实时荧光PCR检测副溶血性弧菌毒力基因方法的建立和应用[J].中国食品卫生杂志,2022,34(1):54-59.

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  • 收稿日期:2021-11-05
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  • 在线发布日期: 2022-03-25
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