梅毒螺旋体荧光定量PCR检测方法的建立

    郭文秀1，云华1，毛兰英1，孙志2，陈宇飞1

    内蒙古国际旅行卫生保健中心，内蒙古呼和浩特 010020；2.宁波出入境检验检疫局

    摘要：目的建立梅毒螺旋体的荧光定量PCR检测方法。方法依据梅毒螺旋体（Treponemapallidum,Tp）DNA多聚酶I(polA)基因序列，设计MGB-Taqman探针和PCR引物，对TPPA法检测到的不同国家和人种梅毒螺旋体抗体阳性患者和阴性人员血液样本进行检测，验证该PCR方法的灵敏度和特异性，以及2种检测方法的一致性。结果PCR产物测序证实新建的荧光PCR方法可以扩增到梅毒螺旋体polA基因区200 bp长DNA片段。该方法特异性强、灵敏度高，可检出每微升血液中10拷贝梅毒DNA。在31例TPPA法检测结果为阳性的样本中，有21例为PCR阳性。证明其中10例TPPA法阳性为非现行感染，可能不具有传染性。统计分析表明该荧光PCR方法与TPPA方法对不同国家和种族人群检测结果未出现显著差异。结论新建的荧光定量PCR方法灵敏度高、特异性好，可排除TPPA法阳性梅毒患者中非现行感染的旅行者，是适用于不同地区和种族的旅行者进行梅毒检测的新方法。

   关键词：梅毒螺旋体，DNA多聚酶I基因，聚合酶链反应，荧光定量聚合酶链反应，MGB-Taqman探针

    中图分类号：R377　文献标识码：B

    Establishment of fluorescence quantitative PCR methodfor

    detection of Treponemapallidum

    GUO Wen-xiu, YUN Hua, MAO Lan-ying, SUN Zhi, CHENYu-fei

    Inner Mongolia International Health CareCenter，HuHot，Inner Mongolia，010020，China

    Abstract:   Objective   To establish afluorescence quantitative PCR method for diagnosis ofTreponemapallidum (Treponemapallidum, Tp).  Methods  Base on the DNA polymerase I gene of Tp, the MGB-Taqman probe andPCR primers were designed and synthesized. The fluorescencequantitative PCR amplification system was optimized. The blood DNAsample of Tp antibody positive patients from different countrieswith different ethnic were detected by the optimizing PCR forsensitivity and specificity evaluation, and the consistency of thenewly built PCR and TPPA diagnosis methods in detection of Tppatients.   Results   PCR amplification products weresequenced and confirmed the 200bp amplified fragment was frompolymerase I gene of Tp. The newly fluorescence quantitative PCRwas specific and sensitive for diagnosis of Tp, which coulld detect10 copies/μl of DNA of Tp in blood. In 31 cases of TPHA positivesamples, only 21 cases were PCR positive. This suggested that therewas no detectable Tp virus in these 10 TPPA positive patients.There were no statistic differences between two diagnostic methods. Conclusion   This newly fluorescence quantitative PCR issensitive and specific for Tp detection, it can exclude thetraveler whom currently not in Tp infectious stage but were TPPApositive. This diagnostic method is suitable to detect Tp fromdifferent countries with different ethnic.

    Key words:   Treponemapallidum, DNA polymerase Igene, Polymerase Chain Reaction；Fluorescence quantitative PCR,MGB-Taqman probe

    《中国国境卫生检疫杂志》2013年10月刊