论文部分内容阅读
定量检测艾滋病患者体内荷毒水平对于评价治疗效果和研究发病机理至关重要 ,我们建立了一种检测 HIV- 1DNA水平的竞争性 PCR方法 ( QC- PCR)。选择 HIV- 1的高度保守区域 gag基因作为靶序列 ,以重组 PCR方法和基因克隆技术构建两种重组质粒 ,分别插入有 gag和 Δ gag片段 ,其中后者比前者内部缺失 50 bp,用作定量检测中的竞争模板。将梯度稀释的竞争模板加入到反应体系中与待测的野生型模板共同扩增 ,PCR产物经 8%聚丙烯凝胶电泳后进行密度扫描测定 ,结果表明该方法稳定可靠 ;8份艾滋病患者的 PBMC DNA标本经该方法的检测也得到较好的结果。我们认为 QC- PCR是一种较为理想的 HIV- 1DNA水平的定量方法 ,非常适合于临床标本的检测和治疗研究的应用。
Quantitative detection of HIV in patients with drug levels in vivo is critical for the evaluation of the therapeutic effect and pathogenesis, we have established a competitive PCR method (QC-PCR) to detect HIV-1 DNA levels. Two highly conserved regions of gag gene of HIV-1 were selected as target sequences. Two recombinant plasmids were constructed by recombinant PCR and gene cloning techniques, respectively, and inserted into the gag and Δgag fragments respectively. The latter was 50 bp shorter than the former and was used as a quantitative The competition template in testing. The gradient dilution competitive template was added to the reaction system to be amplified together with the wild type template to be tested. The PCR product was subjected to density scanning electrophoresis after being electrophoresed on 8% polypropylene gel, and the results showed that the method was stable and reliable; in eight AIDS patients PBMC DNA samples by the method of detection also get better results. We think that QC-PCR is a more ideal quantitative method of HIV-1 DNA level, which is very suitable for the detection and treatment of clinical specimens.