贝类中副溶血弧菌和沙门氏菌荧光定量PCR快速检测方法的建立

Rapid detection of Vibrio parahaemolyticus and Salmonella sp. in shell fishes by Real-time quantitative PCR

为适应进口鲜活水产品的快速检验，有必要建立快速检测多种病原生物的方法。本文针对鲜活水产品中常见的副溶血弧菌和沙门氏菌，设计了特异的荧光引物，建立了检测贝类中这两种细菌的荧光定量PCR体系，并进行了特异性与重复性试验。结果表明，在相同的反应条件下，副溶血孤菌和沙门氏菌均得到了特异性扩增，而阴性对照菌株均未见扩增。副溶血孤菌标准曲线在1．53×10^5～1．53×10^9拷贝／μL之间、沙门氏菌标准曲线在4．89×10^6-4.89×10^10拷贝／μL之间有较好的线性关系。与传统的检测方法相比较，该荧光定量PCR方法检测贝类中的副溶血弧菌和沙门氏菌更为快速准确，结果直观，可以满足口岸进口水产品快速检测的需要。

Rapid detection of pathogens is essential for rapid import inspection of live aquatic animals. In the paper, a real-time quantitative PCR assay Was developed for rapid detection ofV. parahaemolyticus and Salmonella sp. in shellfishes. PCR primers were designed targeting the species-specific gene gyrB of V. parahaemolyticus and the fimY gene of Salmonella sp. The specificity and repeatability of the method were also tested. The results showed that both V. parahaemolyticus strains and Salmonella sp. exhibited typical curves while the other negative control strains were not amplified, indicating the specificity of the assay. The standard curves showed good linearity at the range between 1.53 × 105 and 1.53 ×109 copies/μL for V. parahaemolyticus, and between 4.89 ×106 and 4.89 ×10^10 copies/μL for Salmonella sp. The rapidness and reliability of the assay should be enough for the quick inspection of live imported shellfishes.