清脆的
马铃薯X病毒
放大器
烟草
重组酶聚合酶扩增
亚基因组mRNA
环介导等温扩增
生物
病毒学
烟草花叶病毒
病毒
聚合酶链反应
烟草蚀刻病毒
计算生物学
植物病毒
DNA
遗传学
基因
马铃薯Y病毒
作者
María-Carmen Marqués,Javier Sánchez-Vicente,Raúl Ruiz,Roser Montagud-Martínez,Rosa Márquez-Costa,Gustavo Gómez,Alberto Carbonell,José‐Antonio Daròs,Guillermo Rodrigo
标识
DOI:10.1021/acssynbio.2c00090
摘要
Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread. However, current methods based on the amplification of the viral genome by polymerase chain reaction (PCR) require laboratory conditions. Here, we exploited the CRISPR-Cas12a and CRISPR-Cas13a/d systems to detect three RNA viruses, namely, Tobacco mosaic virus, Tobacco etch virus, and Potato virus X, in Nicotiana benthamiana plants. We applied the CRISPR-Cas12a system to detect viral DNA amplicons generated by PCR or isothermal amplification, and we also performed a multiplexed detection in plants with mixed infections. In addition, we adapted the detection system to bypass the costly RNA purification step and to get a visible readout with lateral flow strips. Finally, we applied the CRISPR-Cas13a/d system to directly detect viral RNA, thereby avoiding the necessity of a preamplification step and obtaining a readout that scales with the viral load. These approaches allow for the performance of viral diagnostics within half an hour of leaf harvest and are hence potentially relevant for field-deployable applications.
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