多药耐药基因QRT-PCR检测中内标准DNA和RNA模板的构建

Construction of the Internal Standard RNA and DNA Templates for MDR1 Quantitative RT-PCR Analysis

利用多药耐药基因(MDR1)全基因质粒和分子克隆技术,经二次克隆将戊型肝炎病毒一段238bP的核苷酸序列插入MDR1 308 bp的目的 cDNA片段,构建了插入突变型MDR1-cDNA重组体作为定量PCR的DNA竞争模板;再经第三次克隆构建了插入突变型MDR1-RNA重组体,最后经体外转录出546 nt的正链RNA作为逆转录的RNA竞争模板。所建立的定量逆转录-多聚酶链反应(QRT-PCR)技术简便、快速、灵敏,可检出1fg水平的MDR1 mRNA,为临床MDR1表达的定量检测提供了确实可行的手段。

1. A 308 bp fragment was amplificated by PCR from the plasmid pHaMDRl/A containing a full-length MDR1 cDNA and cloned reversely into the Sma Ⅰ site of the pUC19 vector, constructing a re-combinant plasmid-pUMDR308. Then, a 238 bp HEV fragment was cut out by EcoRI and BamHI from the plasmid pUHEV and inserted into the Bgl Ⅱ site of pUMDR308, constructing a new recom-binant plasmid-pUMDR308/HEV as an internal DNA competitive template for quantitative PCR. 2. A 546 bp fragment containing MDR1 308 bp and HEV 238 bp was cut out by EcoR I and Xba I from pUMDR308/HEV and cloned into the same enzyme sites of transcriptional vector pSP72, constructing a recombinant plasmid-pSMDR308/HEV. Eventually, pSMDR308/HEV was cleaved with Pvu Ⅱ and transcripted in vitro by T7 RNA polymerase, getting a 546 bp mutant MDR1-RNA positive strand as an internal RNA competitive template for quantitative RT-PCR. 3. 308 bp Target template and 546 bp competitive template were added into the same tube, RT-PCR was carried out. 308 bp and 546 bp PCR products could be distinguished by 1.5% agarose gel electrophoresis. When light degree of both PCR products was same , amount of target mRNA was the same as that of competitive template.