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目的建立副溶血弧菌TaqMan实时-PCR和毒力基因TaqMan双重实时-PCR筛检的实验室检测方法。方法根据副溶血弧菌toxR基因的保守序列设计引物和TaqMan探针,建立检测副溶血弧菌的实时-PCR方法;根据副溶血弧菌耐热直接溶血素(thermostable direct hemolysin,tdh)和耐热相关溶血素(thermostable relatedhemolysin,trh)基因的保守序列设计引物和探针,建立检测致病性副溶血弧菌毒力基因的双重TaqMan实时-PCR方法。对所建立的副溶血弧菌实时-PCR检测方法进行灵敏度和特异度评价。结果副溶血弧菌的检测下限为102拷贝/μl,tdh和trh双重实时PCR的检测下限为102拷贝/μl。针对toxR基因建立的副溶血弧菌实时-PCR方法对11种其他弧菌和肠道细菌的染色体无扩增。结论建立的方法能够特异和敏感地检测副溶血弧菌,并能确定致病性副溶血弧菌的毒力基因,能作为副溶血弧菌的灵敏和快速检测方法。
Objective To establish a real-time PCR-based TaqMan assay for virulence of Vibrio parahaemolyticus and a real-time PCR assay for TaqMan virulence genes. Methods According to the conserved sequence of toxR gene of Vibrio parahaemolyticus, primers and TaqMan probe were designed to establish a real-time-PCR method for detecting Vibrio parahaemolyticus. According to the results of thermostable direct hemolysin (tdh) The primers and probes were designed according to the conserved sequence of the thermostable relatedhemolysin (trh) gene and a double TaqMan real-time PCR method was established to detect virulence genes of pathogenic V. parahaemolyticus. The established Vibrio parahaemolyticus real-time PCR detection sensitivity and specificity of evaluation. Results The detection limit of Vibrio parahaemolyticus was 102 copies / μl, and the detection limit of tdh and trh double real-time PCR was 102 copies / μl. Vibrio parahaemolyticus real-PCR method was established for toxR gene without amplifying the chromosomes of 11 other Vibrio and intestinal bacteria. Conclusion The established method can detect Vibrio parahaemolyticus specifically and sensitively and determine the virulence genes of pathogenic Vibrio parahaemolyticus, which can be used as a sensitive and rapid detection method for Vibrio parahaemolyticus.