Hak Hagit, Ostendorp Steffen, Reza Anton, Ishgur Greenberg Shany, Pines Gur, Kehr Julia, Spiegelman Ziv
Department of Plant Pathology and Weed Research, Agricultural Research Organization - Volcani Institute, Rishon LeZion, Israel.
Department of Biology, Institute of Plant Science and Microbiology, Universität Hamburg, Hamburg, Germany.
J Exp Bot. 2024 Dec 10. doi: 10.1093/jxb/erae495.
Plant viruses are destructive pathogens for various crop species. Rapid, sensitive, and specific detection is crucial for the effective containment of emerging and resistance-breaking viruses. CRISPR/Cas has been established as a new tool for plant virus identification. However, its application for direct detection of viruses in the field is still limited. In this study, we present a CRISPR/Cas13a-based method for rapid detection of different viruses directly from RNA of several crop species, including tomato, cucumber and rapeseed. This method was used to identify the emerging tomato brown rugose fruit virus (ToBRFV), a prominent pathogen in tomato cultivation, and distinguish it from closely related viruses in infected tomato plants. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Finally, we developed a user-friendly, extraction-free, 15-minute protocol for on-site virus detection using a portable fluorescent viewer and a mobile phone camera. This protocol was successfully applied for ToBRFV identification in several commercial greenhouses. These results demonstrate that CRISPR/Cas13a is a robust technology for on-site detection of multiple viruses in different crop plants. This method could be swiftly adapted to identify newly emerging pests, which threaten global food security.
植物病毒是多种作物的毁灭性病原体。快速、灵敏且特异的检测对于有效控制新出现的和能突破抗性的病毒至关重要。CRISPR/Cas已成为用于植物病毒鉴定的一种新工具。然而，其在田间直接检测病毒方面的应用仍然有限。在本研究中，我们提出了一种基于CRISPR/Cas13a的方法，用于直接从几种作物（包括番茄、黄瓜和油菜）的RNA中快速检测不同病毒。该方法用于鉴定新出现的番茄褐色皱纹果病毒（ToBRFV），这是番茄种植中的一种主要病原体，并将其与受感染番茄植株中密切相关的病毒区分开来。在100倍稀释时以及感染早期、病毒症状出现之前就能鉴定出ToBRFV。最后，我们开发了一种用户友好的、无需提取的15分钟方案，使用便携式荧光观察仪和手机摄像头进行现场病毒检测。该方案已成功应用于多个商业温室中的ToBRFV鉴定。这些结果表明，CRISPR/Cas13a是一种用于现场检测不同作物中多种病毒的强大技术。该方法可以迅速调整以鉴定新出现的、威胁全球粮食安全的害虫。
