汉坦病毒S片段荧光定量PCR参考标准品的构建  

Construction of reference standard plasmids of real-time fluorescence quantitative PCR for detecting Hantavirus S segment

作　　者：周欣[1] 李燕婷[2] 沈荣明[2] 沈微娟[2] 瞿涤[1] 

机构地区：[1]复旦大学上海医学院医学分子病毒学教育部重点实验室,上海200032 [2]上海市疾病预防控制中心,上海200336

出　　处：《上海预防医学》2006年第9期432-434,共3页Shanghai Journal of Preventive Medicine

摘　　要：[目的]构建汉坦病毒S片段标准品,用于实时荧光定量PCR检测汉坦病毒。[方法]对S片段基因进行序列分析,利用引物设计软件设计出一对引物和一条荧光探针,提取病人总RNA,逆转录为cDNA,经PCR扩增,产物纯化后与PMD 18-T Vector连接,转化到大肠杆菌DH5α,筛选得到标准品的质粒,提取质粒并进行定量。[结果]重组S片段基因质粒进行荧光定量PCR扩增出现强烈的荧光值增长,表明基因已成功克隆。以不同稀释水平的标准品进行扩增,统计学分析显示C t值与标准品浓度的对数存在良好线性关系,回归系数在0.99以上。成功制备并获得稳定的S片段重组质粒。[结论]该方法能大量制备质粒标准品,如何进行荧光RT-PCR条件的优化是关键所在。[ Objective] To explore the method of preparation for Hantavirus S segment standard plasmids of real -time PCR. [ Methods] Analysis of S gene rearrangement sequence and material from GENEBANK. The primer expression software was used to obtain a couple of primer and fluorescent probe, DNA was extracted from the acute patient, RT - PCR, the PCR product was connected with PMD18 - T vector and then transfered into Ecoil DH5α. The standard recombinant plasmids was gained from the positive plasmids. [ Results] The fluorescence levels were.increased incisively after real -time quantitative PCR amplification, which indicated that S segment was cloned successfully. The Ct value indicated that there was a good linear function in statistics between the Ct value and the concentration gradient of standard DNA specimen. The regression value was 0.99 and over. Steady standard plasmids were obtained. [ Conclusion] This is a good way to get the standard plasmids. How to optimize the condition of real - time PCR is the key.

关 键 词：S片段 实时荧光定量PCR 标准品 

分 类 号：Q78[生物学—分子生物学]