沙门菌、志贺菌和副溶血性弧菌的多重PCR快速检测技术的建立与应用

Establishment and application of multiple PCR technique for the rapid detection of Salmonella sp.,Shigella sp. and Vibrio parahaemolyticus

目的建立快速同时检测沙门菌、志贺菌和副溶血弧菌的多重聚合酶链反应(PCR)技术。方法根据沙门菌hilA基因、志贺菌ipaH基因及副溶血性弧菌tdh基因序列设计引物,进行PCR扩增及电泳检测。同时优化反应体系,测定特异性和灵敏度。结果该多重PCR技术能在8 h内同时检测3种目的菌,通过检测其他9种常见食源性致病菌,结果表明该方法特异性好,多重PCR检测灵敏度分别为:沙门菌104cfu/mL,志贺菌102cfu/mL,副溶血性弧菌104cfu/mL。并进行了人工模拟食品和粪便样品的检测。结论初步建立能在8h内快速、灵敏、特异地同时测定沙门菌、志贺菌和副溶血性弧菌的多重PCR检测技术。

Objective To establish a multiplex PCR technique for the simultaneous detection of Salmonella sp. （SM） , ShigeUa sp. （SP） and Vibrio parahaemolyticus. （VP）. Methods Three sets of primers were designed to amplify the gene segments of hilA of Salmonella sp. , ipaH of Shigella sp. , and tdh of Vibrio parahaemolyticus, and the products were analyzed by electrophoresis. At the same time , this system was optimized , and the specificity and sensitivity of this system were evaluated. Results Three target bacteria were detecte d in 8 h by using this multiple PCR technique. The high specificity was demonstrated by detecting other nine kinds of food-borne pathogenic bacteria, and the detection sensitivity of multiplex PCR were 10^4 cfu/mL for SM, 10^2 cfu/mL for SP, 10^4cfu/mL for VP. Artificially contaminated food and stool samples were inspected with this method. Conclusions A rapid , specific , and sensitive multiplex PCR technique for the simultaneous detection of Salmonella sp. ,Shigella sp. and Vibrio parahaemolyticus, in 8 h has been studied primarily.