摘要
目的建立一种快速、特异、准确定量的单纯疱疹病毒2型（HSV-2）检测方法.方法构建重组质粒pMD18-T-gG作为标准品.以HSV-2的gC基因区为靶基因区,设计合成引物和探针,采用TaqMan MGB技术对HSV-2进行实时定量PGR检测,优化反应体系,并进行方法学评价.结果成功构建重组质粒pMD18-T-gG.建立利用TaqMan MGB技术的real-time PGR方法,其重复性较好[批内CV（变异系数）值为2.29%,批间CV值为4.76%]、特异性较好（对HSV-1、Veto细胞、巨细胞病毒、弓形虫等无特异性扩增）、线性范围好（4.75×10^1～4.75×10^6 copies/μl,r=0.998）,灵敏度达到47.5 copies/μl;与ELISA法进行比较,认为前者的灵敏度更高、检测时间更短.结论建立的TaqMan MGB PGR方法能够快速准确、特异、灵敏地对HSV-2进行定量分析,可为临床诊断提供帮助.
Objective To develop a real-time PCR method for herpes simplex vires-2 （ HST-2）. Methods The recombinant plasmid pMD18-T-gG was constructed as HSV-2 DNA standard for quantitative analysis. According to the principle of TaqMan MGB technique, the primers and probe targeted at HSV-2-gG gene fraction were designed and applied to detect HSV-2. Then the PCR reaction system was optimized and evaluated. Results The recombinant plasmid was constructed successfully. The real-time PCR method for HSV-2 using TaqMan MGB technique was established and the assay showed good reproducibility （intra-assay coefficients of variation was 2.29% and inter-assay coefficients of variation was 4.76% ）, good specificity （it can distinguish HSV-2 from other pathogenic microorganism）, good linear range （4.75 × 10^1 -4.75 × 10^6 copies/μl, r = 0. 998 ） and sensitivity reached 47.5 copies/μl. At the same time, this new established method had more advantages than the ELISA method. Conclusion A novel real-time PCR detecting method for HSV-2 has been developed, which can be used in clinical detection.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第4期375-378,共4页
Chinese Journal of Microbiology and Immunology
基金
南宁市科委攻关项目(南科[2003]27号)
