荧光定量PCR检测梅毒螺旋体方法的建立及初步应用

Establishment and clinical application of fluorescent quantitation PCR for rapid detection of treponema pallidum

目的：采用二聚体蝎型探针技术建立一种高敏感性和特异性的检测梅毒螺旋体（T P ）的荧光定量聚合酶链反应（PCR）方法。方法根据T P特异性外膜蛋白Gpd的基因序列设计引物和荧光探针，采用基因工程技术构建可用于T P定量的标准品，优化荧光定量PCR的反应体系和反应条件，建立检测T P的二聚体蝎型探针定量PCR。采用本研究建立方法和商品化荧光定量PCR试剂盒检测40例临床标本，对比分析检测结果的统计学差异。结果成功构建了T P重组质粒标准品和T P的二聚体蝎型探针定量PCR ，该方法线性范围为101～108 copy／mL ，灵敏度为10 co py／m L ，特异性和敏感性均为100．0％；二聚体蝎型探针定量PC R对疑似梅毒病例阳性检出率显著高于目前商品化Taqman荧光定量PCR试剂盒（82．5％ vs 62．5％，P＜0．05）。结论成功建立了二聚体蝎型探针荧光定量PCR快速检测T P的方法，为临床上T P的早期诊断和防控奠定了基础。

Objective To establish a sensitive and specific fluorescent quantitation PCR （FQ‐PCR） by using duplex scorpion primer for rapid detection of treponema pallidum （TP） .Methods Primer and fluorescent probe were designed based on the gene order of TP outer membrane protein Gpd .Quantitative standard preparation of TP was constructed by using gene recombination .Duplex scorpion primer FQ‐PCR for rapid detection of TP was established by optimizing the reaction system and reaction condition .Totally 40 suspected cases were detected by our FQ‐PCR and commercial FQ‐PCR respectively and the difference was analyzed .Results Duplex scorpion primer FQ‐PCR for rapid detection of TP was established successfully .The linear range of the method was 101 -108 copy/mL and the sensitivity was 10 copy/mL ,susceptibility and specificity were 100 .0% .The positive detection rate of the suspected cases by our method was sharply higher than that by commercial FQ‐PCR kit （82 .5% vs 62 .5% ,P〈0 .05） .Conclu‐sion Duplex scorpion primer FQ‐PCR for rapid detection of TP was established successfully ,which could be used for early diagnosis and preventive control of TP .