以副溶血弧菌毒素调控基因(toxin regulations,toxR)作为靶标基因,设计特异性引物及TaqMan探针,以含toxR基因的质粒为模板,建立质粒拷贝数与CT值的标准曲线,分别采用含toxR基因质粒、纯培养的副溶血弧菌和添加副溶血弧菌的牡蛎(Ostrea)模拟样品进行灵敏度试验,结果表明,其灵敏度分别为15拷贝、18 CFU/mL和180CFU/mL.同一个样品的30次重复性试验表明,试验内及试验间的变异系数分别为0.95%和1.5%.结果显示,本研究建立的副溶血弧菌荧光定量PcR检测方法特异性强、灵敏度高、重复性好,可用于牡蛎等水产品中副溶血弧菌的定量检测.%Vibrio parahaemolyticus (VP) is a pathogen that is the leading cause of shellfish-associated cases of bacterial food poisoning.To establish a real-time PCR for quantitative analysis of toxR gene, a pair of specific primers and TaqMan fluorescent probe of toxR gene of VP were designed.The positive recombinant plasmids of toxR gene served as templates to establish standard curve, which showed corresponding relationship between the copies of plasmids and threshold cycle (CT)values.The lowest limit detection was 15 copies of toxR gene for plasmid, and the sensitivity of pure cultures and simulated oyster sample was 18 CFU/mL and 180 CFU/g, respectively.The coefficient of variation was 0.95％ for intra-assay test and 1.5％ for inter-assay test.One hundred and sixty eight positively established samples were detected from 178 oysters collected from a farm in Guangdong Province.The result showed that the real-time PCR assay for VP had high specificity, sensitivity, repeatability and it was time saving, so it can be used for detecting and monitoring VP in aquatic products.
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