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

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

Plant biotechnology journal 2021 Feb

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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, namely Apple necrotic mosaic virus (ApNMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple 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. © 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Citation

Jian Jiao, Kangkang Kong, Jinmeng Han, Shangwei Song, Tuanhui Bai, Chunhui Song, Miaomiao Wang, Zhenli Yan, Hengtao Zhang, Ruiping Zhang, Jiancan Feng, Xianbo Zheng. Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a-based visual assay. Plant biotechnology journal. 2021 Feb;19(2):394-405