Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, Ljubljana, Slovenia.
J Virol Methods. 2010 Dec;170(1-2):47-56. doi: 10.1016/j.jviromet.2010.08.018. Epub 2010 Sep 9.
A TaqMan(®) one-step reverse transcription real-time PCR (RT-qPCR) assay was developed for the specific detection and relative quantitation of Grapevine fanleaf virus (GFLV), the causal agent of grapevine fanleaf degeneration disease. The assay was targeted to a conservative region located in the 2A(HP) gene of the GFLV RNA2 molecule. The assay specificity was evaluated on GFLV isolates from a wide range of geographical regions and on other viruses infecting grapevines. The sensitivity of the developed assay for GFLV detection was approximately 1000-fold higher than the sensitivity of the conventional ELISA. Concentrations as low as 10 genome copies of GFLV per reaction were reliably detected using RT-qPCR. The new method offers a fast, reliable, specific and sensitive identification test for GFLV that is easily applicable for high-throughput diagnosis of GFLV in different types of grapevine material, including dormant phloem scrapings. The quantitative nature of the assay was evaluated by monitoring the seasonal variation of the amount of GFLV present in the plant phloem.
我们开发了一种 TaqMan(®)一步法逆转录实时 PCR（RT-qPCR）检测方法，用于特异性检测和相对定量葡萄扇叶病毒（GFLV），该病毒是葡萄扇叶退化病的病原体。该检测方法针对 GFLV RNA2 分子 2A(HP)基因中的保守区域。该检测方法的特异性在来自广泛地理区域的 GFLV 分离株以及感染葡萄的其他病毒上进行了评估。该方法检测 GFLV 的灵敏度比传统 ELISA 高约 1000 倍。使用 RT-qPCR 可可靠地检测到每个反应中低至 10 个 GFLV 基因组拷贝的浓度。该新方法提供了一种快速、可靠、特异和敏感的 GFLV 鉴定检测方法，易于应用于不同类型的葡萄材料（包括休眠韧皮部刮取物）中 GFLV 的高通量诊断。通过监测植物韧皮部中 GFLV 含量的季节性变化，评估了该检测方法的定量性质。
