Scuola Normale Superiore, 56126 Pisa, Italy.
J Virol Methods. 2010 May;165(2):161-7. doi: 10.1016/j.jviromet.2010.01.013. Epub 2010 Jan 29.
The aim of this study was the development of gag and pol dual labelled probe real-time PCR and RT PCR assays to quantify the proviral load and the transcripts of the British Visna/maedi virus EV1 strain. Primers and probes were chosen based on the consensus sequences of gag and pol clones representative of EV1 genetic variants. Both PCRs had a detection limit of 3 copies of target gene, with a linearity over 6 orders of magnitude. The performances of the two PCRs in vivo were evaluated and compared on a panel of DNAs extracted from blood of sheep infected experimentally with EV1. The pol assay detected in most cases lower numbers of viral molecules than gag assay, yielding some false negative results. The gag real-time RT PCR had a detection limit of 100 RNA molecules with a linearity over 5 orders of magnitude. This did not result in a lower performance of the RT PCR compared to the PCR in cells permissive for virus replication, which contain higher numbers of viral transcripts than proviral genomes. The real-time assays developed in this study, particularly the gag assay, provide a sensitive tool which can be used to quantify the viral load in experimental infections.
本研究旨在开发 gag 和 pol 双重标记探针实时 PCR 和 RT-PCR 检测方法，以定量测定英国绵羊肺腺瘤病毒 EV1 株的前病毒载量和转录本。根据代表 EV1 遗传变异的 gag 和 pol 克隆的共识序列选择引物和探针。两种 PCR 的检测限均为 3 个拷贝的靶基因，线性度超过 6 个数量级。在一组从感染 EV1 的绵羊血液中提取的 DNA 上，评估了两种 PCR 的体内性能并进行了比较。pol 检测法在大多数情况下检测到的病毒分子数量低于 gag 检测法，导致一些假阴性结果。gag 实时 RT-PCR 的检测限为 100 个 RNA 分子，线性度超过 5 个数量级。这并没有使 RT-PCR 的性能低于对病毒复制有耐受性的细胞中的 PCR，因为这些细胞中的病毒转录本数量高于前病毒基因组。本研究中开发的实时检测方法，特别是 gag 检测法，提供了一种敏感的工具，可用于定量测定实验感染中的病毒载量。
