HIV-1 DNA real-time PCR检测方法的建立及初步应用

Establishment of a real-time PCR assay for quantification of HIV-1 DNA

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 PCR assay for HIV-1 DNA quantification. Methods Two pairs of primers were designed according to the sequence of HIV-1NL4-3LTR gene and human CCR5 gene.A recombinant plasmid containing LTR and CCR5 gene was constructed as a standard control.A real-time PCR assay was established for HIV-1 DNA quantification.Reproducibility and specificity of this assay were also evaluated.HIV-1DNA from 20 HIV-1infected individuals was also detected using this assay. Results A standard control was constructed for HIV-1DNA quantification which showed high linear correlation,the correlation coefficient of the standard curve of both LTR and CCR5 were more than 0.99,and the coefficient of variations were less than 3%.The analytical sensitivity is 1×103 copies/μl.HIV-1DNA from 14 out of 20HIV-1infected individuals were successfully quantified using this assay and the clinical sensitivity reaches70%. Conclusions This real-time PCR assay for HIV-1DNA quantification is rapid,specific and repeatable,which could offer a platform for further study of the correlation between HIV-1DNA and disease progress or evaluation of antiretroviral therapy effect.