Laboratorio de Biología Celular y Retrovirus, CONICET, Hospital de Pediatría Dr. Juan P. Garrahan, Combate de los Pozos 1881, Cuidad Autónoma de Buenos Aires, Argentina.
J Virol Methods. 2010 Dec;170(1-2):160-4. doi: 10.1016/j.jviromet.2010.08.021. Epub 2010 Sep 15.
A real-time quantitative PCR (qPCR) assay using SYBR Green was developed to determine HTLV-I proviral load (pVL) in peripheral blood mononuclear cells (PBMCs), and its performance was evaluated with samples processed as cell lysates and DNA isolated by salting out. Primers targeting the pol region were standardized against the MT2 cell line and HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the albumin gene. The sensitivity, specificity and reproducibility of the qPCR were assessed in the two methods used for DNA processing. The assay had a limit of detection of 400 HTLV-I copies/10(6) PBMCs for both methods, with a broad range of quantitation (2.6log(10) to >5log(10)), and without cross-reactivity with HTLV-II or with HIV-1. The inter- and intra-assay coefficients of variation were less than 2.4%. HTLV-I pVL quantitation in seven blood donor samples processed as either cell lysates or isolated DNA by salting out showed a strong linear correlation and no difference in the calculated pVL (Fisher's exact test, p>0.05). The assay was found to be a low cost, robust and reproducible assay for quantifying HTLV-I pVL in samples processed as cell lysates or as isolated DNA.
建立了一种使用 SYBR Green 的实时定量 PCR (qPCR) 检测方法，用于测定外周血单个核细胞 (PBMC) 中的 HTLV-I 前病毒载量 (pVL)，并通过细胞裂解物处理和盐析分离的 DNA 对其性能进行了评估。针对 pol 区域的引物与 MT2 细胞系进行了标准化，通过定量白蛋白基因将 HTLV-I 拷贝数标准化为细胞 DNA 的量。在用于 DNA 处理的两种方法中评估了 qPCR 的灵敏度、特异性和可重复性。该检测方法对两种方法的检测限均为 400 HTLV-I 拷贝/10(6) PBMC，具有广泛的定量范围（2.6log(10) 至 >5log(10)），并且与 HTLV-II 或 HIV-1 无交叉反应。批内和批间变异系数均小于 2.4%。对作为细胞裂解物或盐析分离 DNA 处理的七个献血者样本中的 HTLV-I pVL 定量表明，计算的 pVL 具有很强的线性相关性且无差异（Fisher 确切检验，p>0.05）。该检测方法被发现是一种经济、稳健且可重复的方法，用于定量处理为细胞裂解物或分离 DNA 的样本中的 HTLV-I pVL。
