副溶血弧菌和溶藻弧菌双重PCR检测方法的建立与初步应用

Establishment and primary application of duplex PCR for detection of Vibrio parahaemolytious and Vibrio alginolyticus

为建立同时快速检测海产品中的副溶血弧菌(VP)和溶藻弧菌(VA)的双重PCR方法,本研究根据VP和VA的toxR基因序列设计针对这两种细菌的两对特异性引物,建立能够快速同时检测这两种细菌的双重PCR方法,并对该反应体系的特异性和灵敏度进行检测。结果显示纯培养细菌VP和VA的检测灵敏度分别为2.32×103cfu/mL和2.56×103cfu/mL,临床病料检测灵敏度分别为2 cfu和3 cfu;与枸橼酸杆菌、沙门氏菌、美人鱼弧菌、霍乱弧菌、麦氏弧菌无交叉反应。研究表明本实验方法操作简便快速、特异性强、灵敏度高、稳定性好,并且经济实惠,值得推广应用。

In this study, a duplex PCR （dPCR） was established and optimized to detect Vibrio parahaemolytious （VP） and Vibrio alginolyticus （VA） simultaneously, two sets of specific primers were designed according to the toxR sequence of VP and VA available in GenBank. The results showed that the detection limits of the dPCR assay were 2.32 × 10^3 cfu/mL and 2.56× 10^3 cfu/mL for VP and VA with pure bacteria culture, and 2 cfu and 3 cfu for VP and VA with clinic samples, respectively. The method possessed a better specificity and no cross-reaction with other enterobacteria such as Citro Bacter, Salmonella, V. damsela, V. cholera, V. mets-chnikovii. The method could provide a useful assay for identification and inspection of VP or VA from fishery product in import and export.