小鼠脑脊髓炎病毒非编码蛋白片段实时荧光定量PCR标准品的构建

doi:10.3969/j.issn.1674-5817.2012.01.002

实验动物与比较医学 ›› 2012, Vol. 32 ›› Issue (1): 8-13.DOI: 10.3969/j.issn.1674-5817.2012.01.002

• 第11届中南地区实验动物科技交流会优秀论文选刊 • 上一篇下一篇

小鼠脑脊髓炎病毒非编码蛋白片段实时荧光定量PCR标准品的构建

袁文, 张钰, 王静, 刘香梅, 黄韧

广东省实验动物重点实验室, 广东省实验动物监测所, 广州 510260

收稿日期:2011-10-20 出版日期:2012-02-25 发布日期:2012-02-25
作者简介:袁文(1979-),男,助理研究员,研究方向:实验动物病毒分子生物学,E-mail:ywen010101@163.com
基金资助:
广东省科技计划项目(2007A060305003); 广东省实验动物重点实验室(2007B060101002)

Construction of Reference Standard Plasmids of Real-time Quantitative PCR for Detecting Theiler's Murine Encephalomyelitis Virus UTR Segment

YUAN Wen, ZHANG Yu, WANG Jin, LIU Xiang-mei, HUANG Ren

Guangdong Key Laboratory of Laboratory Animals, Guangdong Laboratory Animals Monitoring Institute, Guangzhou 510260, China

Received:2011-10-20 Online:2012-02-25 Published:2012-02-25

摘要/Abstract

摘要： 目的构建小鼠脑脊髓炎病毒(TMEV)非编码蛋白(UTR)片段标准品,用于实时荧光定量PCR检测小鼠脑脊髓炎病毒。方法 RT-PCR 扩增TMEV UTR片段上80~1094 nt之间长度为1 014 bp的片段,将目的片段连接至pMD18-T载体,转化至DH5α感受态细胞。分别经PCR鉴定和序列测定验证重组质粒。分光光度计测量重组质粒的吸光值,换算成拷贝数浓度后作10倍梯度稀释制得质粒标准品。然后进行实时荧光定量PCR分析, 绘制标准曲线。结果 TMEV UTR片段成功克隆至pMD18-T载体中, 测序结果表明重组质粒中插入的UTR序列正确, 实时荧光定量PCR分析10倍梯度系列稀释的质粒标准品所得到的标准曲线良好,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。结论成功构建了TMEV UTR 片段实时荧光定量PCR标准品,为今后TMEV实时荧光定量PCR检测打下了基础。

关键词: 小鼠脑脊髓炎病毒, 实时荧光定量PCR, 标准品

Abstract: Objective To construct a recombinant plasmid as the standard for Theiler's murine encephalomyelitis virus (TMEV)detection by real-time quantitative PCR. Methods A specific TMEV untranslated region (UTR) fragment in size of 1014 bp was amplified by RT-PCR, The PCR product was ligated with pMD18-T vector and transformed into E.coli DH5α. Recombinant plasmid was identified by PCR and sequencing. The concentration of recombinant plasmid was analyzed through its absorption at 260 nm. Standard curve was constructed by real- time quantitative PCR with ten-fold serial dilutions of recombinant plasmid. Results The fragment of UTR was successfully cloned into pMD18-T vector. The quantitative standard curve of recombinant standard plasmids was obtained, statistics analysis indicated that there was a good linear correlation between the Ct value and the concentration gradient of standard plasmids. The regression value was over 0.99. Conclusion The standard plasmid for detecting TMEV with real-time PCR has been constructed successfully.

Key words: Theiler's murine encephalomyelitis virus (TMEV), Real-time PCR, Standard plasmid

中图分类号:

R332
Q95-33

袁文, 张钰, 王静, 刘香梅, 黄韧. 小鼠脑脊髓炎病毒非编码蛋白片段实时荧光定量PCR标准品的构建[J]. 实验动物与比较医学, 2012, 32(1): 8-13.

YUAN Wen, ZHANG Yu, WANG Jin, LIU Xiang-mei, HUANG Ren. Construction of Reference Standard Plasmids of Real-time Quantitative PCR for Detecting Theiler's Murine Encephalomyelitis Virus UTR Segment[J]. Laboratory Animal and Comparative Medicine, 2012, 32(1): 8-13.

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小鼠脑脊髓炎病毒非编码蛋白片段实时荧光定量PCR标准品的构建

Construction of Reference Standard Plasmids of Real-time Quantitative PCR for Detecting Theiler's Murine Encephalomyelitis Virus UTR Segment

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