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目的建立同时检测副溶血性弧菌毒力基因tdh和trh的双重荧光PCR方法。方法筛选副溶血性弧菌tdh和trh毒力基因的特异性引物和探针,建立基于TaqMan探针双重荧光PCR扩增体系,进行特异性、灵敏度实验,并对本实验室保存的54株副溶血性弧菌进行tdh、trh毒力基因的检测,以了解不同来源的副溶血性弧菌携带毒力基因的状况。结果建立的双重荧光PCR方法特异度强(100%),最低检测浓度达到20 cfu/ml,对本实验室保存的副溶血性弧菌进行毒力基因的检测结果显示,从临床分离的10株副溶血性弧菌和海产品样品中分离的1株副溶血性弧菌均为tdh扩增阳性,trh扩增阴性。结论所建立的方法特异性好,灵敏度高,适用于副溶血性弧菌的毒力基因检测。
Objective To establish a dual-fluorescence PCR method for simultaneous detection of virulence genes tdh and trh of Vibrio parahaemolyticus. Methods The specific primers and probes of virulence genes of Vibrio parahaemolyticus tdh and trh were screened, and the double fluorescent PCR amplification system based on TaqMan probe was established for specificity and sensitivity test. 54 strains of parahemolytic Vibrio parahaemolyticus tdh, trh virulence genes detected in order to understand the different sources of Vibrio parahaemolyticus carrying virulence genes. Results The double-fluorescent PCR method was highly specific (100%) and the lowest detection concentration was 20 cfu / ml. Virulence genes of Vibrio parahaemolyticus preserved in our laboratory showed that 10 isolates Vibrio parahaemolyticus and seafood samples isolated from a Vibrio parahaemolyticus are positive tdh amplification, trh negative amplification. Conclusion The established method has good specificity and high sensitivity and is suitable for virulence gene detection of Vibrio parahaemolyticus.