Université catholique de Louvain, Earth and Life Institute, Applied Microbiology - Phytopathology, Croix du Sud, 2/3, B-1348 Louvain-la-Neuve, Belgium.
J Virol Methods. 2010 Nov;169(2):385-90. doi: 10.1016/j.jviromet.2010.08.010. Epub 2010 Aug 17.
The 3'-end region of many virus isolates has been shown to possess conserved sequences in addition to the presence of numerous genomic and subgenomic RNAs. Utilizing these sequences, a broad-spectrum reverse transcription-polymerase chain reaction protocol has been developed to detect all the known Indian peanut clump virus and Peanut clump virus isolates, that cause peanut clump diseases in West Africa and India. The primers were targeted at the highly conserved 3'-untranslated regions of the PCV RNA-1 and RNA-2. The conservation was confirmed by sequencing these untranslated regions of RNA-1 for six isolates and RNA-2 for one isolate. The conserved structure of the RNA-1 and RNA-2 was observed and the importance of this region for the virus survival was confirmed. The primers were also designed for virus quantitation using a Taqman(®)-based real-time RT-PCR. The use of RT-PCR and real-time quantitative RT-PCR improved the sensitivity of PCV detection compared to ELISA. RT-PCR also led to the detection of IPCV and PCV on two new natural hosts: Oldenlandia aspera and Vigna subterranea. Real-time RT-PCR is considered to be an ideal tool for identifying resistant sources to both IPCV and PCV.
许多病毒分离株的 3'末端区域除了存在大量基因组和亚基因组 RNA 外，还具有保守序列。利用这些序列，开发了一种广谱逆转录-聚合酶链反应方案，以检测所有已知的印度落花生丛矮病毒和落花生丛矮病毒分离株，这些病毒会导致西非和印度的落花生丛矮病。引物针对的是 PCV RNA-1 和 RNA-2 的高度保守 3'非翻译区。通过对 6 个分离株的 RNA-1 和 1 个分离株的 RNA-2 的非翻译区进行测序，证实了这种保守性。观察到 RNA-1 和 RNA-2 的保守结构，并证实了该区域对病毒存活的重要性。还为使用 Taqman（®）基于实时 RT-PCR 的病毒定量设计了引物。与 ELISA 相比，RT-PCR 和实时定量 RT-PCR 的使用提高了 PCV 的检测灵敏度。RT-PCR 还导致在两个新的天然宿主：Oldenlandia aspera 和 Vigna subterranea 上检测到 IPCV 和 PCV。实时 RT-PCR 被认为是鉴定对 IPCV 和 PCV 均具有抗性的来源的理想工具。
