Highly sensitive methods based on seminested real-time reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced RNA and proviral DNA
Highly sensitive methods based on seminested real-time reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced RNA and proviral DNA
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DOI：
10.1128/jcm.00055-08
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发表时间：
2008-07-01
影响因子：
9.4
通讯作者：
Lukashov, Vladimir V.
中科院分区：
文献类型：
--
作者：
Pasternak, Alexander O.;Adema, Karen W.;Lukashov, Vladimir V.
高效抗逆转录病毒疗法（HAART）是治疗1型人类免疫缺陷病毒（HIV - 1）感染的标准疗法，其疗效通过测量血浆中的病毒RNA载量来评估。当血浆中的HIV - 1载量保持在商业检测方法的检测限以下时，患者被视为治疗成功。然而，在接受HAART治疗的患者中，病毒复制和进化仍在继续，这最终可能导致耐药性HIV - 1毒株的产生和治疗失败。为了监测外周血单个核细胞（PBMC）中的这种低水平病毒复制，需要灵敏的方法来测量HIV - 1分子标记物。我们报道了用于定量检测PBMC中未剪接和多重剪接的HIV - 1 RNA以及前病毒DNA的高灵敏度方法的开发。这些方法基于创新的半巢式实时逆转录 - 聚合酶链反应（RT - PCR），它结合了实时PCR的准确性和精确性以及巢式PCR的灵敏度。我们表明，新开发的方法在灵敏度、准确性、动态范围以及对临床样本中HIV - 1 RNA和DNA的定量检测能力方面优于传统的单步实时RT - PCR。这些易于操作的方法可广泛用于研究，包括临床研究，以监测病毒复制的细胞内过程。
The effectiveness of highly active antiretroviral therapy (HAART), the standard of care for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, is assessed by measuring the viral RNA load in plasma. A patient is considered to be successfully treated when the HIV-1 load in plasma stays below the detection limit of commercial assays. However, virus replication and evolution do continue in patients under HAART, which may eventually result in the development of drug-resistant HIV-1 strains and therapy failure. To monitor this low-level virus replication in peripheral blood mononuclear cells (PBMC), sensitive methods are required to measure HIV-1 molecular markers. We report the development of highly sensitive methods for the quantitation of unspliced and multiply spliced HIV-1 RNA and proviral DNA in PBMC. The methods are based on innovative seminested real-time reverse transcription-PCR (RT-PCR) that combines the accuracy and precision of real-time PCR and the sensitivity of nested PCR. We show that the newly developed methods are superior to the conventional single-step real-time RT-PCR in their sensitivity, accuracy, dynamic range, and the power of quantitative detection of HIV-1 RNA and DNA in clinical samples. These easy-to-perform methods can be widely used in research, including clinical studies, to monitor intracellular processes of virus replication.