副溶血性弧菌实时荧光定量PCR标准模板的构建和检测方法的建立

Construction of Standard Template and Development of Real-Time Fluorescence Quantitative PCR Assay for Detection of Vibrio parahemolyticus

目的:构建肉制品副溶血性弧菌实时荧光定量PCR的标准阳性模板和检测方法。方法:以副溶血性弧菌toxR基因上特异性片段为目标,设计并合成引物及Taqman探针,将目标片段连接到PGM-T载体上构建重组质粒,建立实时荧光定量检测体系,并考察方法的灵敏性、特异性、重复性和准确性。结果:构建出副溶血性弧菌荧光定量PCR检测方法的标准模板并建立了相应的检测方法,获得的标准曲线方程为:Y=-3.151 lgX+42.86,灵敏度40copies/反应体系,对副溶血性弧菌具有特异性,同时,该方法具有良好的重复性,变异系数小于5%。结论:使用基于Taqman探针技术的荧光定量PCR检测方法能够对食品中致病性副溶血性弧菌进行快速、简便、准确、高效的定量检测。

Objective： To construct recombinant plasmids for use as standard positive template and establish a real-time （RT） fluorescent quantitative PCR assay for determining Vibrio parahemolyticus in meat products. Methods： Primers and Taqman probes were designed and synthesized using the specific fragment of toxRS gene from Vibrio Parahemolyticus as target sequence. Recombinant plasmids were constructed by inserting the target gene into PGM-T vector. ART fluorescent quantitative PCR assay was established and its sensitivity, specificity, repeatability and accuracy were investigated. Results： Recombinant plasmids were successfully constructed for use as standard positive template for fluorescent quantitative PCR. The following calibration curve was established： Y =--3.151 lgX ＋ 42.86. The sensitivity of the PCR assay was 40 copies per reaction system, and good specificity for Vibrio parahemolyticus and high repeatability were observed. The intra-batch and inter-batch coefficients of variation for five replicate assays were both below 5%. Conclusion： The fluorescent quantitative PCR based on Taqman probes allows rapid, simple, accurate and efficient detection of Vbrio parahemolyticus in foods.