PDF(Pubmed)
Abstract：
Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.
摘要：
由木薯褐条病毒(CBSV)和乌干达木薯褐条病毒(UCBSV)惹起的木薯褐条病(CBSD)是木薯最重要的经济病毒性疾病。由于木薯是营养繁殖的作物，快速和灵敏诊断的发展将有助于识别无病毒种植材料和制定有效的管理策略。在这项研究中,一个快速的，开发了特异性和灵敏的实时逆转录重组酶聚合酶扩增（RT-RPA）测定法，用于实时检测CBSV和UCBSV。RT-RPA能够检测到从受感染的木薯叶获得的仅为2pg/µl的纯化RNA，灵敏度相当于通过定量实时逆转录PCR（qRT-PCR）获得的灵敏度，在37°C下20分钟内Further,RT-RPA直接从粗叶和茎提取物中检测到每种目标病毒，避免繁琐和昂贵的高质量RNA分离。开发的RT-RPA测定法提供了一种有价值的诊断工具，可用于木薯种子认证和病毒抗性育种计划，以确保向农民分发无病毒木薯种植材料。这是关于开发和验证基于粗树液的RT-RPA测定法以检测木薯植物中的木薯棕色条纹病毒（UCBSV和CBSV）感染的第一份报告。
