汉坦病毒S片段荧光定量PCR参考标准品构建.docVIP

汉坦病毒S片段荧光定量PCR参考标准品构建摘要： ［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Ct 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。 关键词：S片段； 实时荧光定量PCR； 标准品；中图分类号：R 372 文献标识码： Ａ Construction of reference standard plasmids of real-time fluorescence quantitative PCR for detecting Hantavirus S segmentZHOU Xin?1, LI Yan-ting?2, SHEN Rong-ming?2, SHEN Wei-juan?2, QU Di?1 (1.Key Laboratory of Medical MolecuLar Virology Ministry of Education and Health-Shanghai Medical College, Fudan Unversity,Shanghai 200032, China; 2.Shanghai Municipal Center for Disease Control and Prevention, Shanghai200336, China) Abstract: ［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 Ssegment 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. Key words: S segment； Real-time PCR； Standard plasmids 近几年来，实时荧光定量PCR技术因高精确度，高灵敏性，污染少等优点已在临床医学及生物医学基础科研中得到广泛应用。它在每个循环后都对扩增产物进行检测，其灵敏度比传统PCR高出2～3个数量