实时荧光PCR快速检测水产品中副溶血性弧菌的研究

目的建立快速检测水产品中副溶血性弧茵的实时荧光PCR方法。方法利用副溶血性孤菌TDH基因的保守序列设计引物和探针,建立快速检测副溶血性弧菌的实时荧光PCR方法。结果本研究建立的检测副溶血性弧菌的实时荧光PCR方法,其纯茵检测低限低于10cfu/PCR反应体系;对模拟阳性样品直接检测,检测低限为10~3cfu/g。模拟样品经增茵培养4～6h后,检测低限达1cfu/g;用本法对24株标准菌株/参考菌株进行检测,结果除副溶血性弧菌呈阳性外,其他23株非副溶血性弧菌均呈阴性反应;重复性试验结果:批内和批间变异系数均小于1%。结论本研究建立的副溶血性弧菌实时荧光PCR方法具有快速、灵敏、特异和重复性好的优点,可在8h内对水产品中的副溶血性弧菌进行定性或定量检测。

Study on Rapid Detection of Vibrio parahaemolytichus in Aquatic Products by Real-Time PCR

Objective To develop a real-time PCR for the rapid detection of Vibrio parahaemolytichus in aquatic products. Methods A real-time PCR was developed for the rapid detection of Vibrio parahaemolytichus based on the primers and probe which were designed for the conservative domain of TDH gene. Results A real-time PCR for the rapid detection of Vibrio parahaemolytichus was developed.The quantitative detection limit of this method was less than 10cfu/PCR reaction for pure cultured broth, and 103cfu/g and 1 cfu/g for non-cultured and 4h-6h- cultured artificial contaminated aquatic products respectively. In the specificity test, among 24 reference/standard strains, only one strain of Vibrio parahaemolytichus was positive, while 23 strains of non- Vibrio parahaemolytichus were all negative. All of the coefficient of variation of intra-assay and inter-assay were less than 1%. Conclusion The real-time PCR is a repeatable, specific , sensitive and rapid method for the detection of Vibrio parahaemolytichus. It takes less than 8h to qualitatively or quantitatively detect Vibrio parahaemolytichus in aquatic products.