TaqMan双重荧光PCR用于快速检测模拟临床标本中产毒型O139群霍乱弧菌的研究  

Multiplex real-time TaqMan-based PCR method for rapid detection of toxigenic Vibrio cholerae Serogroup O139 in simulated clinical specimens

作　　者：徐丹戈[1] 黄世旺[1] 方叶珍[1] 徐昌平[2] 张政[2] 包芳珍[1] 李剑[1] 蒋雪凤[1] 卢亦愚[2] 

机构地区：[1]杭州市江干区疾病预防控制中心,杭州310004 [2]浙江省疾病预防控制中心,杭州310051

出　　处：《中国卫生检验杂志》2011年第4期901-904,共4页Chinese Journal of Health Laboratory Technology

基　　金：杭州市医药卫生科技计划项目(2006B079)

摘　　要：目的:研究建立TaqM an双重荧光PCR技术,用于快速检测模拟临床标本中产毒型O139群霍乱弧菌。方法:以O139群霍乱弧菌O抗原编码基因rfb O139和毒力基因CT的特异性序列设计引物和TaqM an探针,建立优化TaqM an双重荧光PCR反应体系,进行特异性与敏感性的研究,并应用于模拟粪便感染和外环境监测标本的快速检测中。结果:该方法对产毒型O139群霍乱弧菌检测具有高度特异性,对rfbO139和CT基因序列检出限达到1.0×102cfu/m l或5 cfu/PCR反应体系,对模拟粪便感染和外环境监测标本的检测结果与实际情况完全一致,整个检测过程最快仅需3 h。结论:本研究建立的产毒型O139群霍乱弧菌双重荧光PCR检测技术具有特异性好、敏感性高、快速易操作等优点,可用于临床粪便标本和外环境监测样本的快速检测。Objective：To establish a TaqMan-based multiplex real-time PCR assay for rapid detection of toxigenic Vibrio cholerae Serogroup O139 in simulated clinical specimens.Methods：The gene sequences of Cholera toxin and specific O antigen biosynthetic gene rfb O139 Serogroup were aligned using the biologic software and the specific primers and probe were designed in the conserved region of the CT and rfb O139 gene for Vibrio cholerae serogroup O139.The reaction conditions were optimized and the sensitivity,specificity of the assay were evaluated.The simulated clinical specimens prepard from the fecal and the environment were detected by this assay.Results：For specific detecting the toxigenic Vibrio cholera Serogroup O139,the detection limits of the assay for rfb O139 and CT gene all were 100 cfu/ml（or 5 cfu/PCR reaction）.The result of detecting the simulated clinical specimens prepard from the fecal and the environment was in accord with actual situation.It took only three hours to do the multiplex real-time PCR.Conclusion：This assay is a rapid,sensitive and specific one for the detection of toxigenic Vibrio cholerae Serogroup O139 from fecal and environment specimens.

关 键 词：霍乱弧菌 双重荧光PCR CT rfb O139 

分 类 号：R378.3[医药卫生—病原生物学]