Osman Fatima, Leutenegger Christian, Golino Deborah, Rowhani Adib
Department of Plant Pathology, University of California, Davis, CA 95616, USA.
J Virol Methods. 2007 Apr;141(1):22-9. doi: 10.1016/j.jviromet.2006.11.035. Epub 2007 Jan 12.
Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Grapevine Leafroll associated viruses 1-5 and 9 (GLRaV-1-5 and -9). The assays were evaluated against a wide range of geographically distributed isolates. Geographical locations included South Africa, Europe, Australia, Asia, Latin America and the United States. Sequences were piled up from the most conserved regions of these geographically diverse isolates and TaqMan primers and probes were designed, targeting the regions with 100% sequence identity. Improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control to validate the quality of the extracted RNA proved to generate better diagnostic assays. The real-time TaqMan RT-PCR assays were compared to the conventional RT-PCR assays for the detection of viruses using purified total RNA as well as crude extract. The data showed that when using total RNA extracted either by the Qiagen RNeasy method or by an ABI automated system more isolates were detected in comparison to crude extract. The optimum volume of crude extract prepared in GES for use in real-time TaqMan RT-PCR cocktail was determined to be 1 microl per reaction. In addition this report showed that TaqMan RT-PCR was more sensitive than conventional one-step RT-PCR for testing different isolates of these viruses either using RNA or crude tissue extract.
已开发出实时逆转录聚合酶链反应（TaqMan）检测方法，用于特异性检测葡萄卷叶相关病毒1 - 5和9（GLRaV - 1 - 5和 - 9）。针对广泛地理分布的分离株对这些检测方法进行了评估。地理位置包括南非、欧洲、澳大利亚、亚洲、拉丁美洲和美国。从这些地理分布多样的分离株的最保守区域堆积序列，并设计TaqMan引物和探针，靶向具有100%序列同一性的区域。改进RNA提取技术，并使用18S核糖体RNA TaqMan检测作为RNA特异性内部对照来测试RNA质量，以验证提取的RNA质量，这被证明能产生更好的诊断检测方法。将实时TaqMan逆转录聚合酶链反应检测方法与传统逆转录聚合酶链反应检测方法进行比较，以使用纯化的总RNA以及粗提物来检测病毒。数据表明，与粗提物相比，当使用Qiagen RNeasy方法或ABI自动化系统提取的总RNA时，检测到的分离株更多。确定在GES中制备用于实时TaqMan逆转录聚合酶链反应混合物的粗提物的最佳体积为每个反应1微升。此外，本报告表明，对于使用RNA或粗组织提取物检测这些病毒的不同分离株，TaqMan逆转录聚合酶链反应比传统的一步逆转录聚合酶链反应更灵敏。
