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

Abstract

Summary 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.

LAMP-Coupled CRISPR–Cas12a Module for Rapid and Sensitive Detection of Plant DNA Viruses

“…However, despite the various applications of CRISPR technology in plant virology [17][18][19][20][21][22], CRISPR-based diagnostic techniques are rarely used for plant pathogens. A recent study demonstrated the successful use of CRISPR-based diagnostics for the sensitive, specific detection of plant RNA viruses and viroids in apple (Malus domestica) [23]. This technique and the current platform lay the foundation for further development of CRISPR-based diagnostics for the rapid, sensitive, specific detection of (in principle) all plant pathogens.…”

CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet

“…In addition, for confirmatory checking of the RT-RPA product, optimized conditions for product visualization and downstream sequence analysis revealed the legitimacy of the RT-RPA product, thus validating the method for BNYVV detection. Previously developed molecular methods for BNYVV detection include regular RT-PCR, nested RT-PCR, and IC-RT-LAMP assays (Henry et al, 1995;Morris et al, 2001;Almasi and Almasi, 2017). However, these methods require gel electrophoresis for downstream product analysis, which hinders high-throughput application.…”

“…Enzymelinked immunosorbent assay (ELISA), a protein-based detection technology, has been used for many years for field soil evaluations because of the ease of implementation and availability of reagents commercially (Torrance et al, 1988). Later, molecular biologybased standard reverse transcription (RT)-PCR and nested RT-PCR methods were developed to increase the sensitivity of BNYVV detection (Henry et al, 1995;Morris et al, 2001). An immunocapture loop-mediated isothermal amplification (IC-RT-LAMP) method subsequently was reported to be more sensitive (Almasi and Almasi, 2017).…”

Differential Detection of the Tobamoviruses Tomato Mosaic Virus (ToMV) and Tomato Brown Rugose Fruit Virus (ToBRFV) Using CRISPR-Cas12a

“…Recently, CRISPR/Cas12a was applied for the detection of RNA [ 43 , 44 ] and DNA [ 45 ] plant viruses, suggesting its potential application in the detection of plant viral diseases. Here we adopt the CRISPR/Cas12a technology to specifically identify the emerging ToBRFV and to distinguish it from a closely related tobamovirus, ToMV.…”