A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load
A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load
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DOI：
10.1038/nprot.2008.108
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Lusso, Paolo
中科院分区：
文献类型：
--
作者：
Malnati, Mauro S.;Scarlatti, Gabriella;Lusso, Paolo
人类免疫缺陷病毒1型（HIV - 1）前病毒DNA的定量越来越多地用于测量HIV - 1细胞储存库，这是评估HIV - 1感染者抗逆转录病毒治疗方案疗效的一个有用标志物。此外，前病毒DNA载量是围产期HIV - 1感染早期诊断的一个特定标志物，并且可能独立于血浆HIV - 1 RNA水平和CD4(+) T细胞计数预测HIV - 1疾病进展。HIV - 1高度的遗传变异性对设计一种能够以相似效率检测主要（M）HIV - 1组内所有基因亚型的通用定量检测方法构成了严峻挑战。在此，我们描述了一种高度灵敏的实时PCR方案，与其他方法相比，该方案能够以更高的准确度对几乎所有M组HIV - 1毒株进行正确定量。该方案包括三个阶段，即DNA提取/裂解、细胞DNA定量和HIV - 1前病毒载量评估。由于PCR设计的稳健性，该检测可在粗细胞提取物上进行，因此它可能适用于即使是发展中国家的临床样本的常规分析。在采血后1天内即可实现对HIV - 1前病毒载量的准确定量。
Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1-infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4(+) T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.