摘要
目的:制备用于nm23-H1基因mRNA实时荧光定量PCR检测的质粒标准品。方法:通过PCR扩增出目的片断,纯化后T-A连接至pMD18-T载体,转化宿主菌E.coliDH5α,获得阳性克隆;通过PCR扩增、双酶切鉴定和测序分析,确认重组质粒完整正确;大量抽提重组质粒,测定其拷贝浓度,10倍稀释成梯度标准品,并进行荧光定量PCR检测分析。结果:nm23-H1基因目的片段成功重组至pMD18-T载体上,获得的重组质粒保持了目的片段的特异性和序列完整性。梯度浓度标准质粒的荧光定量PCR结果显示,循环阈值(C t)与起始模板量的对数值之间有着良好的线性关系。结论:成功构建了nm23-H1基因实时荧光定量PCR质粒标准品。
Objective：To construct recombinant plasmid pMD18 ～ nm23 - H1/p as the standards for nm23 - H1 gone detection by real -time fluorescence quantitative PCR. Methods：A 253 bp DNA segment of nm23 - H1 gone cDNA was amplified by PCR and T -A cloned into pMD18 -T vector, then transformed into bacterium DHSα. The recombinant plasmid DNA extracted from positive clones was identified by PCR amplification, digestion with restriction cndonuclcases Sal Ⅰ and EcoR Ⅰ, and sequence dctcrmination. The concentration of purified plasmid DNA solution was determined by analyzing its absorption at 260nm, and then ten -fold serially diluted into 10^8 to 10^2 copy/μl. Standard quantitative curves were constructed by real -time PCR detection with the series of plasmid standards. Results：The target segment was successfully recombined into pMD18 - T vector with correct sequences. The results of real - time fluorescence quantitative PCR with the series of plasmid standards showed an ideal linear relationship between the logarithmic values of initial template concentration and cycle threshold values. Condusion：A series of recombined plasmid standards for nm23 -H1 gone real -time PCR analysis have been successfully constructed.
出处
《中国卫生检验杂志》
CAS
2007年第4期604-605,615,共3页
Chinese Journal of Health Laboratory Technology
基金
中国博士后科学基金资助项目(20060390737)
广东省科技计划项目(2005B50301017)
关键词
nm23-HI基因
实时荧光定量PCR
重组质粒
标准品
nm23 -H1 gone
Real-time fluorescence quantitative PCR
Recombinant plasmid
Reference standards
