Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.
J Virol Methods. 2012 Jan;179(1):38-44. doi: 10.1016/j.jviromet.2011.09.016. Epub 2011 Sep 24.
Two TaqMan-based real-time One-Step RT-PCR assays were developed for the rapid and efficient detection of Raspberry bushy dwarf virus (RBDV) and Raspberry leaf mottle virus (RLMV), two of the most common raspberry viruses in North America and Europe. The primers and probes were designed from conserved fragments of the polymerase region of each virus and were effective for the detection of different isolates tested in this study. The RBDV assay amplified a 94bp amplicon and was able to detect as few as 30 viral copies. Whereas the RLMV assay amplified a 180bp amplicon and detected as few as 300 viral copies from plant and aphid RNA extracts. Both assays were significantly more sensitive than their corresponding conventional RT-PCR methods. The sensitivity of the RLMV assay was also tested on single aphids after a fixed acquisition access period (AAP). In addition, the assays revealed a novel synergistic interaction between the two viruses, where the concentration of RBDV was enhanced ∼400-fold when it occurred in combination with RLMV compared to its concentration in single infections. The significance of this finding and the importance of the development of real-time RT-PCR assays for the detection of RBDV and RLMV are discussed.
两种基于 TaqMan 的实时一步 RT-PCR 检测方法被开发出来，用于快速、高效地检测覆盆子布什矮化病毒（RBDV）和覆盆子叶片斑驳病毒（RLMV），这两种病毒是北美和欧洲最常见的覆盆子病毒。引物和探针是根据每个病毒聚合酶区域的保守片段设计的，对本研究中测试的不同分离株均有效。RBDV 检测方法扩增了 94bp 的扩增子，能够检测到低至 30 个病毒拷贝。而 RLMV 检测方法扩增了 180bp 的扩增子，能够从植物和蚜虫 RNA 提取物中检测到低至 300 个病毒拷贝。这两种检测方法的灵敏度都明显高于相应的常规 RT-PCR 方法。还在固定的取食获得期（AAP）后对单个蚜虫进行了 RLMV 检测方法的灵敏度测试。此外，该检测方法还揭示了两种病毒之间存在一种新的协同相互作用，与单独感染相比，当 RBDV 与 RLMV 同时存在时，其浓度增强了约 400 倍。讨论了这一发现的意义以及开发用于检测 RBDV 和 RLMV 的实时 RT-PCR 检测方法的重要性。
