Laboratorio de Virus Linfotrópicos Humanos-Retrovirus HIV-HTLV, Instituto de Virología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Enfermera Gordillo s/n, Ciudad Universitaria, CP: X5000HUA Córdoba, Argentina.
J Virol Methods. 2013 May;189(2):383-7. doi: 10.1016/j.jviromet.2013.02.018. Epub 2013 Mar 7.
A quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detect and quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers were designed, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral load was normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, the qPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assay reproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutions of the standard was 2.97 copies. The assay had an excellent dynamic range from 10⁵ to 10¹ copies per reaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performance of the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers ranged from 2.2×10² to more than 8.3×10⁴ copies/10⁶ PBMCs. The high sensitivity and wide dynamic range allowed the determination of a broad range of HTLV-1 proviral loads in infected individuals. This assay is a valuable alternative diagnostic tool when current available serological assays are insufficient. In addition, it will facilitate the study of the relationship between proviral load and pathogenesis.
建立了一种使用 SYBR Green 染料的实时荧光定量 PCR（qPCR）检测方法，用于检测和定量外周血单个核细胞（PBMCs）中的 HTLV-1 前病毒 DNA。设计了引物，并对该检测方法进行了标准化，以扩增新型保守的 HTLV-1 tax 区。通过定量人白蛋白基因来将前病毒载量标准化为细胞 DNA 的量。首先，通过使用标准品稀释液进行 PROBIT 分析来评估 qPCR 的特异性、灵敏度、动态范围以及内和间试验重复性。通过 PROBIT 分析确定的检测限为 2.97 拷贝。该检测方法具有从 10⁵到 10¹拷贝/反应的优异动态范围和良好的内和间试验重复性，CV 值小于 2%。其次，在 40 名 HTLV-1 血清阳性个体中测试了 qPCR 的性能。HTLV-1 携带者的前病毒载量范围为 2.2×10²至超过 8.3×10⁴拷贝/10⁶ PBMCs。高灵敏度和宽动态范围允许在感染个体中确定广泛的 HTLV-1 前病毒载量。当当前可用的血清学检测方法不足时，该检测方法是一种有价值的替代诊断工具。此外，它将有助于研究前病毒载量与发病机制之间的关系。
