三重real-time PCR检测副溶血弧菌主要毒力基因

针对编码副溶血弧菌的特异性基因和主要毒力基因tlh、tdh、ureR进行引物及探针设计，通过优化反应条件，建立基于Taqman探针快速检测副溶血弧菌毒力基因的三重实时聚合酶链式反应（real-time polymerase chain reaction，real-time PCR）方法。利用10 株副溶血弧菌和22 株非副溶血弧菌对设计的引物及探针进行特异性验证，结果表明设计的引物及探针具有很高的特异性；优化后的引物浓度分别为tdh 0.15 μmol/L、tlh 0.15 μmol/L、ureR 0.80 μmol/L，探针浓度分别为HEX 0.50 μmol/L、FAM 0.50 μmol/L；该方法即使在高浓度的背景细菌存在下，DNA浓度与Ct值均呈良好的线性关系，检出限为1.8×102 拷贝/mL；以完整菌细胞的悬液为模板时，预变性时间为30 min，扩增检测效果与以基因组DNA（gDNA）为模板相当（ΔCt＜1）。本研究建立的三重real-time PCR方法能实现快速定量检测副溶血弧菌，并能有效区分致病性及非致病性副溶血弧菌，为副溶血弧菌的快速定量检测和风险评估提供快速、灵敏、准确的方法。

Establishment of a Triplex Real-time PCR Method for Detecting the Main Virulence Genes of Vibrio parahaemolyticus

In this study, in order to establish a triplex real-time polymerase chain reaction (real-time-PCR) method based on a Taqman probe for the rapid detection of the virulence genes of Vibrio parahaemolyticus, primers and probes were designed targeting V. parahaemolyticus-specific genes and its main virulence genes tlh, tdh and ureR, and the reaction conditions were optimized. Ten strains of V. parahaemolyticus and 22 non-V. parahaemolyticus strains were used to verify the specificity of the designed primers and probes. The results showed that the primers and probes presented high specificity. The optimized primer concentrations were 0.15, 0.15 and 0.80 μmol/L for tdh, tlh and ureR, respectively, and the optimized probe concentrations were 0.50 μmol/L for both HEX and FAM. Even at a high concentration of background bacteria, this method exhibited a good linear relationship between DNA concentration and Ct value, and the lowest detection limit was 1.8 × 102 copies/mL. When the pre-denaturation time was set to 30 min, the amplification result using the intact bacterial cell suspension as the template was comparable to that using genomic DNA (gDNA) (ΔCt < 1). The triplex real-time PCR method can not only allow for rapid and quantitative detection of V. parahaemolyticus, but also effectively distinguish between pathogenic and non-pathogenic V. parahaemolyticus, thus providing a rapid, sensitive and accurate method for risk assessment of pathogenic V. parahaemolyticus.