以toxR-S为靶基因的副溶血弧菌PCR检测方法的建立

Development of a PCR for detection of Vibrio parahaemolyticus based on toxR-S gene sequence

目前,副溶血弧菌PCR检测方法大多基于毒力基因或看家基因,存在假阳性和漏检的风险,建立敏感而特异的快速分子检测方法迫在眉睫。本研究在对toxRS基因序列进行系统发育树构建及同源性分析的基础上,发现toxR、toxS基因均有作为种特异性检测靶基因的可能,故分别设计引物Primers-R、Primers-S及Primers-R-S,通过优化PCR反应条件建立了相应的PCR检测方法。通过对57株副溶血弧菌和19株亲缘相近菌及食源性致病菌的检测,toxR-SPCR显示出较好的稳定性和特异性,检测极限达2pg基因组DNA。对359份市场海产品样品的比对检测结果表明,toxR-S PCR与国标(GB4789.7-2013)规定的方法的符合率高达95.82%,敏感性100%,特异性92.39%。对两种方法检测时阳性不一致的15份样品,以其总细菌DNA为模板扩增副溶血弧菌看家基因;测序比对结果显示,这15份样品与副溶血弧菌同源性高达97%以上。上述结果表明,本研究建立的toxR-S PCR具有快速、特异、敏感等优点,可被广泛用于副溶血弧菌的快速检测。

Several PCR assays based on virulence or housekeeping genes had been developed for the identification of Vibrio parahaemolyticus.But they have a risk in high probability of miss detection and false positive detection.In this study,three pairs of PCR primers（Primers-S,Primers-Rand Primers-R S）were designed based on the phylogenetic and homologous relationships of toxRand toxSgenes of Vibrio genus.Specificity tests had showed that the Primers-R-S had higher stability and species-specificity than the other two pairs of primers.Therefore,the toxR-S PCR based on Primers-R-S was refined as the rapid V.parahaemolyticus detection assay.The detection limitation of the toxR-S PCR was 2pg genomic DNA.Comparative analysis between toxR-S PCR and the classical method in GB4789.7-2013 was performed in359 seafood samples,which resulted in 100% sensitivity,92.39% specificity and 95.82% coincidence rate.The above-mentioned results confirmed that toxR-S PCR was specific,rapid,and sensitive,and it could be widely applied to rapid identification of V.parahaemolyticus isolates and seafood samples.