Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a-based visual assay

文献类型： 外文期刊

第一作者： Jiao, Jian

作者： Jiao, Jian;Kong, Kangkang;Han, Jinmeng;Song, Shangwei;Bai, Tuanhui;Song, Chunhui;Wang, Miaomiao;Feng, Jiancan;Zheng, Xianbo;Yan, Zhenli;Zhang, Hengtao;Zhang, Ruiping

作者机构：

关键词： apple viral diseases; LbCas12a; RT-RPA; visualized detection; in-field application

期刊名称：PLANT BIOTECHNOLOGY JOURNAL （ 影响因子：9.803； 五年影响因子：9.555 ）

ISSN： 1467-7644

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页码：

收录情况： SCI

摘要： Co-infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a-based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namelyApple necrotic mosaic virus(ApNMV),Apple stem pitting virus(ASPV),Apple stem grooving virus(ASGV),Apple chlorotic leaf spot virus(ACLSV) andApple scar skin viroid(ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide-conjugated gold nanoparticles. The CRISPR/Cas12a-RT-RPA platform exhibited comparable sensitivity to RT-qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT-PCR detection for 52 samples. This novel Cas12a-based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.

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