水产品中创伤弧菌和副溶血弧菌双重PCR检测方法的建立

Establishment of duplex PCR method for detection of Vibrio vulnificus and Vibrio parahaemolyticus in aquatic products

目的建立一种同步检测创伤弧菌和副溶血弧菌的双重PCR方法。方法选择副溶血弧菌tlh基因和创伤弧菌vvhA基因作为靶序列各设计一对引物。用合成的引物对副溶血弧菌和创伤弧菌进行双重PCR扩增,确定特异性和最低检出限。然后用此方法对53株副溶血弧菌和7株创伤弧菌进行检测。结果确定了双重PCR检测创伤弧菌和副溶血弧菌的最优反应条件,其中退火温度为60℃,方法具有较好的特异性。对副溶血弧菌的最低限为1.0×10~2 CFU/mL,创伤弧菌最低限为4.2×10~4 CFU/mL。双重PCR对分离株检测符合率达100%。结论建立的双重PCR方法简便、快速、特异性好,可同时检测副溶血弧菌和创伤弧菌,为水产品中病原菌的基层检测提供解决方案。

Objective To develop a duplex polymerase chain reaction(PCR)method for rapid detection of both Vibrio vulnificus and Vibrio parahaemolyticus in aquatic products.Methods A pair of primers was designed as target sequence for each of the tlh gene of Vibrio parahaemolyticus and vvhA gene of Vibrio vulnificus.The duplex PCR amplification was performed on Vibrio parahaemolyticus and Vibrio vulnificus with the synthetic primers,and the specificity and minimum detection limits were determined.Then the 53 isolates of Vibrio parahaemolyticus and 7 isolates of Vibrio vulnificus were tested using this developed method.Results The optimum parameters of duplex PCR for detection of Vibrio vulnificus and Vibrio parahaemolyticus were determined.The annealing temperature was 60℃,and the method has good specificity.The minimum for detecting Vibrio parahaemolyticus was 1.0×10~2 CFU/mL,and the minimum for detecting Vibrio vulnificus was 4.2×10~4 CFU/mL.The consistent rate of duplex PCR for isolates was 100%.Conclusion The established duplex PCR is a simple,rapid and specific method for detecting synchronously Vibrio parahaemolyticus and Vibrio vulnificus.It can provide a solution for the detection of pathogens in aquatic products.