摘要
为实现水产食品中多种弧菌同步快速检测,拟建立一种同时检测副溶血弧菌、溶藻弧菌和霍乱弧菌的三重PCR快速检测方法。根据副溶血弧菌的bla CARB-17基因、溶藻弧菌的gyrB基因和霍乱弧菌的toxR基因设计特异性引物。针对3种弧菌分别建立单重PCR体系并优化各项主要反应参数,确定最佳退火温度。然后建立多重PCR体系并优化其反应参数,分析三重PCR的特异性和灵敏度;并验证其实效性。结果表明:三重PCR的最佳退火温度为60℃,能同时扩增出副溶血弧菌bla CARB-17基因的230 bp片段、溶藻弧菌gyrB基因的337 bp片段,霍乱弧菌toxR基因的130 bp片段,灵敏度分别为0.583 ng/μL、0.394 ng/μL、0.866 ng/μL。该三重PCR检测方法可以从实际样品中快速特异地检出三种弧菌,具有较好的特异性,较高的灵敏度,为实际生产中检测水产食品中的弧菌污染提供参考。
In order to realize the synchronous and rapid detection of a variety of Vibrio,a triple PCR rapid detection method was developed to simultaneously detect Vibrio parahaemolyticus,Vibrio alginolyticus and Vibrio cholerae in aquatic foods.Specific primers were designed according to blaCARB-17 gene of Vibrio parahaemolyticus,gyrB gene of Vibrio alginolyticus and toxR gene of Vibrio cholerae.The single PCR system was established for the three types of Vibrio,and the reaction parameters were optimized to determine the optimal annealing temperature.Then,the multiplex PCR system was established and the reaction parameters were optimized to analyze the specificity and sensitivity of the triple PCR method,and the method effectiveness was verified.The results showed that the optimum annealing temperature of triple PCR detection method was 60℃,and the 230 bp fragment of blaCARB-17 gene of Vibrio parahaemolyticus,337 bp fragment of gyrB gene of Vibrio alginolyticus,and 130 bp fragment of toxR gene of Vibrio cholerae could be amplified at the same time.The sensitivities for Vibrio parahaemolyticus,Vibrio alginolyticus and Vibrio cholerae were 0.583 ng/μL,0.394 ng/μL and 0.866 ng/μL,respectively.Three kinds of Vibrio can be detected quickly and specifically in actual samples,which showed that the triple PCR detection method had good practicability,it was fast and efficient,had good specificity and high sensitivity.The triple PCR detection method can provide reference for Vibrio contamination in aquatic food production.
作者
周茜
方林芳
李凤霞
陈清森
胡元庆
ZHOU Xi;FANG Linfang;LI Fengxia;CHEN Qingsen;HU Yuanqing(School of Biological Science and Biotechnology,Minnan Normal University,Zhangzhou 363000)
出处
《中国食品添加剂》
CAS
2024年第4期262-270,共9页
China Food Additives
基金
福建省自然科学基金面上项目(2021J01995)
福建省现代农业生猪产业技术体系(2020-2022)。
关键词
副溶血弧菌
溶藻弧菌
霍乱弧菌
多重PCR
快速检测
Vibrio parahaemolyticus
Vibrio alginolyticus
Vibrio cholerae
multiple PCR
rapid detection
