Rapid on-site detection of crop RNA viruses using CRISPR/Cas13a

清脆的 植物病毒 作物 生物 病毒 番茄斑萎病毒属 生物技术 作物保护 病毒学 农学 遗传学 基因 番茄斑点萎蔫病毒
作者
Hagit Hak,Steffen Ostendorp,Anton Reza,Shany Ishgur Greenberg,Gur Pines,Julia Kehr,Ziv Spiegelman
出处
期刊:Journal of Experimental Botany [Oxford University Press]
标识
DOI:10.1093/jxb/erae495
摘要

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.
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