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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。
Objective To establish a dual real-time fluorescence PCR method for Vibrio parahaemolyticus and Vibrio vulnificus. Methods The primers and probes of tdh and vvh A gene were designed after the Gen Bank database was searched for sequences and aligned. The annealing temperature, primer, probe, Mg2 +, Taq DNA polymerase and dNTPs concentration in PCR reaction were optimized, and the reaction system and reaction conditions were determined. The specificity and sensitivity of the new method were also analyzed and the simulated samples were detected . Results Double real-time PCR was used to detect Vibrio parahaemolyticus and Vibrio vulnificus. The specificity of this method is good, with a detection limit of 103 CFU / ml. Conclusion The established dual-fluorescence PCR method can detect Vibrio parahaemolyticus and Vibrio vulnificus rapidly, sensitively and specifically.