Alberta Plant Health Lab, Crop Diversification Centre North, Alberta Agriculture and Forestry, Edmonton, AB, Canada.
Crop Diversification Centre South, Alberta Agriculture and Forestry, Brooks, AB, Canada.
PLoS One. 2020 Jun 24;15(6):e0230403. doi: 10.1371/journal.pone.0230403. eCollection 2020.
Tomato brown rugose fruit virus (ToBRFV) is a member of Tobamovirus infecting tomato and pepper. Within North America, both the United States and Mexico consider ToBRFV to be a regulated pest. In Canada, the presence of ToBRFV has been reported, but an efficient diagnostic system has not yet been established. Here, we describe the development and assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect ToBRFV. The LAMP test was efficient and robust, and results could be obtained within 35 min with an available RNA sample. Amplification was possible when either water bath or oven were used to maintain the temperature at isothermal conditions (65°C), and results could be read by visual observation of colour change. Detection limit of the LAMP was eight target RNA molecules. Under the experimental conditions tested, LAMP was as sensitive as qPCR and 100 times more sensitive than the currently used RT-PCR. We recommend this sensitive, efficient LAMP protocol to be used for routine lab testing of ToBRFV.
番茄褐色皱果病毒（ToBRFV）是一种感染番茄和辣椒的烟草花叶病毒。在北美，美国和墨西哥都将 ToBRFV 视为受管制的有害生物。在加拿大，已报告存在 ToBRFV，但尚未建立有效的诊断系统。在这里，我们描述了一种基于环介导等温扩增（LAMP）的检测 ToBRFV 的方法的开发和评估。LAMP 试验高效且稳健，可在 35 分钟内从可用的 RNA 样本中获得结果。当使用水浴或烤箱将温度维持在等温条件（65°C）时，扩增是可能的，并且可以通过观察颜色变化来读取结果。LAMP 的检测限为 8 个靶 RNA 分子。在测试的实验条件下，LAMP 与 qPCR 一样敏感，比目前使用的 RT-PCR 敏感 100 倍。我们建议将这种敏感、高效的 LAMP 方案用于常规实验室检测 ToBRFV。
