Quantification of Human Immunodeficiency Virus Type 1 Proviral Load by a TaqMan Real-Time PCR Assay

Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log(10). Reproducibility was evaluated in intra- and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 10(6) PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 10(6) PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy (P = 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5′ end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4(+) cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.

人类免疫缺陷病毒1型（human immunodeficiency virus type 1, HIV-1）前病毒DNA可作为探索病毒储存库及监测抗逆转录病毒治疗的有效标志物，尤其当血浆中HIV-1 RNA检测不到时。本研究开发了一种采用TaqMan实时荧光定量PCR（TaqMan real-time PCR）技术的检测方法，可实现HIV-1前病毒DNA的定量检测。该检测方法对5拷贝的HIV-1前病毒DNA可实现100%的检测灵敏度，且检测线性范围覆盖6个log₁₀量级。本研究通过对携带HIV-1前病毒基因组的8E5细胞系进行独立提取，在批内和批间实验中评估了检测的重复性：两组的变异系数（coefficients of variation, CV）分别为13%和27%；同时对1例平均前病毒载量为每10⁶外周血单个核细胞（peripheral blood mononuclear cells, PBMC）26拷贝的患者的外周血单个核细胞进行检测，其批内和批间变异系数分别为46%和56%。一项横断面研究对21例患者的PBMC前病毒载量进行检测，结果显示其中位载量为每10⁶ PBMC 215拷贝，检测范围为<10至8381拷贝。在一项纵向研究中，16例原发性感染患者接受1年抗逆转录病毒治疗后，其中15例的前病毒载量出现显著下降（P=0.004）。剩余1例患者的HIV-1前病毒DNA可被检测出，但无法准确定量，原因在于其TaqMan探针5'端存在点突变。研究未观察到前病毒载量与血浆中CD4⁺细胞计数或HIV-1 RNA水平存在相关性。TaqMan实时荧光定量PCR检测方法灵敏度高，可适配大批量样本的检测需求。如今，将HIV-1前病毒DNA监测应用于患者的常规临床监测，即可充分明确该指标的全部应用价值。