副溶血弧菌TaqMan双重实时-聚合酶链反应检测方法的建立

Establishment of Vibrio parahaemolyticus detection by duplex TaqMan real-time PCR assay

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 real-time PCR TaqMan assay for the detection of Vibrio parahaemolyticus and duplex real-time PCR TaqMan assay for the detection of toxic genes of V.parahaemolyticus.MethodsThe conserved region of toxR gene of V.parahaemolyticus was used to design primers and TaqMan probe,and the real-time PCR TaqMan system to detect V.parahaemolyticus was established.The conserved region of tdh and trh genes were used to design primers and probes,and the duplex real-time PCR TaqMan system to detect toxic genes of pathogenic V.parahaemolyticus was established.The sensitivity and specificity of the assays to detect V.parahaemolyticus was evaluated.ResultsThe sensitivity of the Real-time PCR TaqMan system to detect V.parahaemolyticus was 10^2 copies/μl;and the sensitivity of the duplex TaqMan Real-time PCR system to tdh and trh was 10^2 copies/μl.No amplification was observed in the templates of other bacterium when they were detected by the assay targeting toxR gene of V.parahaemolyticus.Conclusion The established assays can detect V.parahaemolyticus sensitively and specifically and determine toxic genes of pathogenic V.parahaemolyticus,so it is a sensitive and specific way to detect V.parahaemolyticus.