CAJ | 학술논문

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。
목적건립대부용혈성호균（Vibrio parahaemolyticus）특이성검측 toxR（과막전록격활단백）기인화tdh（열은정성직접용혈소）독력기인적Taqman탐침쌍색형광PCR검측방법。방법근거부용혈성호균toxR기인화tdh기인，분별설계인물화탐침，건립Taqman탐침쌍색형광PCR확증체계，진행특이성、령민도시험；대부용혈성호균분리균주실시검측，료해기tdh기인화tdh기인분포정황。결과결과표명，부용혈성호균표준균주화3주종식물중독환자중분리획득적분리주균출현toxR기인화tdh확증곡선，이용조호균、단증리사특균등31주호균속기타균주화장간균과적균주미견확증곡선。종식품중분리적37주부용혈성호균분리주균미휴대tdh독력기인。부용혈성호균검측령민도가체도3.6×102 cfu/mL。결론해방법가용우동시검측식품중부용혈성호균적특이성화독력기인。
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 (transmembrane 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 amplifica-tion system. The optimized method was used to detect the isolated bacterial strains and to explore the distribu-tion 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. However, 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.