双重实时荧光PCR检测副溶血性弧菌和创伤弧菌方法的建立

目的建立双重实时荧光PCR检测副溶血性弧菌和创伤弧菌的方法。方法在Gen Bank数据库查找序列并比对后设计tdh和vvh A基因引物和探针。对PCR反应退火温度、引物、探针、Mg2+、Taq DNA聚合酶及d NTPs浓度进行优化,确定反应体系和反应条件,并对新建方法的特异性、灵敏度等方面进行分析,对模拟样品进行检测。结果建立了双重实时荧光PCR检测副溶血性弧菌和创伤弧菌的方法。该方法的特异性较好,检测限为103 CFU/ml。结论建立的双重荧光PCR方法可快速、灵敏、特异地检测副溶血性弧菌和创伤弧菌。

Development of a duplex real-time PCR assay for detection of Vibrio parahaemolyticus and Vibrio vulnificus

Objective To develop a duplex real-time PCR assay for detection of Vibrio parahaemolyticus and Vibrio vulnificus. Methods The sequences of tdh and vvh A genes were searched and blasted, primers and probes of these two genes were designed. The annealing temperature and the concentration of primers, probes, Mg^2＋, Taq DNA polymerase and d NTPs were optimized for detection. The specificity and sensitivity of the new method were analyzed. The simulative samples were tested. Results A real-time method for detection of V. parahaemolyticus and V. vulnificus was developed. This method showed good specificity and the detection limit was 10^3 CFU / ml. Conclusion This duplex real-time PCR assay can rapidly, sensitively and specifically identify V. parahaemolyticus and V. vulnificus.