水产品中副溶血弧菌、溶藻弧菌和霍乱弧菌三重PCR检测方法的建立OACSTPCD

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.