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目的建立对副溶血性弧菌(Vibrio parahaemolyticus)特异性检测toxR(跨膜转录激活蛋白)基因和tdh(热稳定性直接溶血素)毒力基因的Taqman探针双色荧光PCR检测方法。方法根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102cfu/mL。结论该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。
Objective To establish a Taqman-based two-color fluorescent PCR method for the specific detection of toxR (transmembrane transcriptional activator) gene and tdh (thermostable hemolysin) virulence genes in Vibrio parahaemolyticus. Methods According to toxR gene and tdh gene of Vibrio parahaemolyticus, primers and probes were designed respectively, and Taqman probe dual-color fluorescent PCR amplification system was established for specificity and sensitivity test. Vibrio parahaemolyticus isolates were tested to understand The tdh gene and tdh gene distribution. The results showed that the standard strains of Vibrio parahaemolyticus and three strains isolated from food poisoning patients showed toxR gene and tdh amplification curve, and Vibrio alginolyticus, Listeria monocytogenes and other 31 strains of Vibrio Strains belonging to other strains and Enterobacteriaceae showed no amplification curve. The 37 isolates of Vibrio parahaemolyticus isolated from food did not carry tdh virulence genes. Vibrio parahaemolyticus detection sensitivity can reach 3.6 × 102cfu / mL. Conclusion The method can be used for simultaneous detection of Vibrio parahaemolyticus in food specific and virulence genes.