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目的建立HIV-1DNA的实时荧光定量聚合酶链反应(real-time PCR)检测方法。方法根据HIV-1NL4-3LTR基因及人CCR5基因序列设计引物,构建质粒标准品,建立绝对定量检测方法,对检测方法的重复性和特异性进行评价。用该方法对20例HIV-1感染者HIV-1DNA进行定量检测。结果构建了HIV-1DNA绝对定量的标准品质粒,标准曲线的相关系数在0.99以上,重复性试验中变异系数小于3%,检测下限为1×103拷贝/μl。20例HIV-1感染者样品中,14例样品检测到HIV-1DNA,检出率为70%。结论 HIV-1DNA real-time PCR检测方法具有快速、特异性强和稳定性好等优点,为进一步研究HIV-1DNA与疾病进程的关系和评价抗病毒疗效等方面奠定了基础。
Objective To establish a real-time fluorescence quantitative polymerase chain reaction (real-time PCR) assay for HIV-1 DNA. Methods Based on the sequences of HIV-1NL4-3LTR gene and human CCR5 gene, primers were designed to construct plasmid standard. The method of absolute quantification was established and the repeatability and specificity of the method were evaluated. The method was used to detect the HIV-1 DNA in 20 HIV-1 infected patients. Results The absolute quantitative standard plasmid of HIV-1 DNA was constructed. The correlation coefficient of the standard curve was above 0.99. The coefficient of variation of the reproducibility test was less than 3%. The detection limit was 1 × 103 copies / μl. Of the 20 HIV-1 infected samples, HIV-1 DNA was detected in 14 samples and the detection rate was 70%. Conclusion The detection of HIV-1 DNA by real-time PCR is rapid, specific and stable. It lays the foundation for further research on the relationship between HIV-1 DNA and disease progression and evaluation of anti-viral efficacy.