食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立

Detection of Vibrio parahaemolyticus toxR and tdh genes in foods by dual-color fluorescent real-time PCR

中文关键词:  副溶血性弧菌  toxR基因  tdh基因  双色荧光PCR  检测

英文关键词:Vibrio parahaemolyticus  toxR gene  tdh gene  dual-color fluorescent real-time PCR  detection

中文摘要:

      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3.6×102 cfu/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。

英文摘要:

      Objective To establish a novel method to specifically detect Vibrio parahaemolyticus toxR and tdh genes in food by dual-color fluorescent real-time PCR. Methods The primers and probes targeting the toxR (trans-membrane transcription activator) and tdh (thermostable direct hemolysin) were designed, and their specificity and sensitivity were tested with the Taqman probe dual-color fluorescent real-time PCR amplification system. The optimized method was used to detect the isolated bacterial strains and to explore the distribution of toxR and tdh genes. Results Amplification curves of both toxR and tdh genes were obtained from standard V. parahaemolyticus strains and the three V. parahaemolyticus strains isolated from food-poisoning patients. How-ever, no such amplification curve was observed for all the other newly isolated 31 bacterial strains, including Vibrio alginolyticus and L. monocytogenes from the genus Vibrio and the family Enterobacteriaceae. All the 37 food V. parahaemolyticus strains did not carry the tdh virulence gene. Furthermore, the detection sensitivity of this novel method reached 3.6×102 cfu/mL. Conclusion The novel dual-color fluorescent real-time PCR method was able to specifically and effectively detect V. parahaemolyticus in food samples.