多重PCR快速检测5种重要致病性弧菌

Multiplex PCR assay for rapid detection of five important pathogenic vibrios

目的建立一种快速检测霍乱弧菌、副溶血性弧菌、创伤弧菌、拟态弧菌和溶藻弧菌5种重要致病性弧菌的多重PCR方法。方法以霍乱弧菌omp W基因、副溶血性弧菌toxR基因、创伤弧菌vvh A基因、拟态弧菌VMH基因和溶藻弧菌gyr B基因为靶基因设计特异性引物,优化建立多重PCR反应体系,系统评价其特异性和检测下限值。结果成功构建致病性弧菌多重PCR检测方法。评价结果显示其特异性为100%,霍乱弧菌、副溶血性弧菌和拟态弧菌的检测下限值为100 CFU/ml,创伤弧菌和溶藻弧菌的检测下限值为1 000 CFU/ml;多重PCR的弧菌检出率明显高于传统分离培养方法(56%vs 17%)。结论该多重PCR方法快速、灵敏、特异性好,可用于海环境和水产品中致病性弧菌的监测。

Objective To establish a rapid multiplex PCR assay for simultaneous detection of five important pathogenic vibrios,including Vibrio cholerae,Vibrio parahaemolyticus,Vibrio vulnificus,Vibrio mimicus and Vibrio alginolyticus. Methods Firstly specific primers were designed according to the conserve regions in omp W gene of V. cholerae,toxR gene of V. parahaemolyticus,vvh A gene of V. vulnificus,VMH gene of V. mimicus,and gyr B gene of V. alginolyticus. Then the multiplex PCR assay was established after various optimization. And finally specificity and limit of detection of the assay were analyzed. Results The multiplex PCR assay for pathogenic vibrios detection was established successfully. The specificity was 100% and the limit of detection for V. cholerae,V. parahaemolyticus,V. mimicus was 100 CFU / ml,while that for V. vulnificus and V. alginolyticus was 1 000 CFU / ml. The overall detection rate of multiplex PCR assay was significantly higher than that of the traditional culture methods（ 56% vs 17%）. Conclusion The multiplex PCR assay is a rapid,sensitive and specific method which would play an important role in the inspection and detection of pathogenic vibrios in the marine environment and aquatic products.