Department of Plant Pathology, National Chung Hsing University, Taichung 40227, Taiwan.
PhD Program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taichung 40227, Taiwan.
Plant Dis. 2021 Oct;105(10):2867-2872. doi: 10.1094/PDIS-12-20-2730-RE. Epub 2021 Oct 26.
A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are listed as quarantine pathogens in many countries. Among them, columnea latent viroid, pepper chat fruit viroid, potato spindle tuber viroid, tomato apical stunt viroid, tomato chlorotic dwarf viroid, and tomato planta macho viroid are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from 1 fg to 10 ng, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR, but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate the ability to screen a large number of solanaceous plants and seeds intended for import and export.
许多病毒和类病毒感染茄科植物，导致严重的产量损失。一些种子传播的类病毒在许多国家被列为检疫病原体。其中，黄瓜隐潜病毒、辣椒聊天果病毒、马铃薯纺锤块茎类病毒、番茄顶端生长迟缓病毒、番茄黄化矮缩病毒和番茄普拉塔马乔病毒是主要关注的对象。本研究旨在设计和测试通用引物，以便使用一步法逆转录 PCR（RT-PCR）和逆转录环介导等温扩增（RT-LAMP）检测茄科植物中的六种类病毒。结果表明，一对简并引物可用于一步法 RT-PCR 从茄科种子和植物中扩增六种 Pospiviroids。此外，设计了五个引物并用于 RT-LAMP 来扩增六种 Pospiviroids。成功检测所需的最小类病毒 RNA 浓度因类病毒和检测方法的不同而有所差异，范围从 1 fg 到 10 ng。一般来说，RT-LAMP 比 RT-PCR 更敏感，但两种检测方法都是快速和高度敏感的工具，可用于检测六种 Pospiviroids。目前用于这些类病毒的检测方法至少需要两组不同的引物。本研究中开发的检测方法可以促进对大量进口和出口的茄科植物和种子进行筛选的能力。
