San Diego Center for AIDS Research, University of California San Diego, La Jolla, California, USA.
Veterans Medical Research Foundation, San Diego, California, USA.
J Clin Microbiol. 2018 Nov 27;56(12). doi: 10.1128/JCM.01158-18. Print 2018 Dec.
We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with qPCR results ( = 0.7052; = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.
我们利用脉冲场凝胶电泳（PFGE）从感染 HIV 的细胞中纯化高分子量 DNA。这种纯化方法与我们之前描述的液滴数字 PCR（ddPCR）检测法相结合，开发了一种方法，可以定量检测无低分子量 HIV DNA 的前病毒整合 HIV DNA。逆转录病毒 DNA 中的 2 长末端重复（2-LTR）环完全从 HIV DNA 样本中清除。从相同样本开始，完整检测的技术重复在经过长期抗逆转录病毒治疗（ART）后，对 HIV 感染患者细胞中的细胞 DNA 中整合的 HIV 序列进行定量，没有统计学差异。PFGE-ddPCR 检测法与最常用于测量前病毒整合 HIV DNA 的定量 PCR（qPCR）检测法进行了比较。Spearman 非参数相关确定 PFGE-ddPCR 结果与 qPCR 结果呈正相关（ = 0.7052； = 0.0273）。总之，PFGE-ddPCR 是一种灵敏、可重复且强大的方法，可用于测量前病毒整合 HIV DNA，并且在理论上比以前描述的检测法更准确，因为它是整合 HIV DNA 的直接测量方法。
