One-Step Reverse-Transcription Recombinase-Aided Amplification CRISPR/Cas12a-Based Lateral Flow Assay for Fast Field Screening and Accurate Differentiation of Four Major Tobamoviruses Infecting Tomato and Pepper

Abstract

Several tobamoviruses cause substantial economic losses to tomato and pepper crops globally, especially the pepper mild mosaic virus (PMMoV), tomato brown rugose fruit virus (ToBRFV), tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV). A fast and accurate detection method is essential for virus identification. An all-in-one reaction method combining a one-step reverse-transcription recombinase-aided amplification (RT-RAA) and CRISPR/Cas12a-based lateral flow assay in one mixture was developed to rapidly screen and accurately differentiate among these four tobamoviruses for field detection in tomato and pepper plants. With a generic RT-RAA primer set and a mix of four specific crRNAs, along with a portable metal incubator and the use of a crude extraction method, this method screened for PMMoV, ToBRFV, ToMV, and ToMMV concurrently in less than 1 h, enabling field workers to take action immediately. The accurate differentiation of these four viruses could be achieved by later adding a single specific crRNA.

Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses

“…By incorporating specially modified probe primers into the amplification system and collaborating with LFT, RPA-LFT has emerged as a primary method for diagnosing plant viruses with varied genome types, encompassing +ssRNA, negative sense (−) ssRNA, ambisense RNA (±RNA), dsRNA, ssDNA, dsDNA, and even viroids with naked circle RNA [1,7,[18][19][20][21][22][23][24][25][26][27][28][29][30] (Table 1). Rice stripe mosaic virus -ssRNA 30 min 10 7 dilution [38] Tomato brown rugose fruit virus +ssRNA 20 min 10 1 copies /reaction [39] Pepper mild mottle virus +ssRNA <1 h - [40] Tomato mosaic virus [40] Tomato mottle mosaic virus [40] *: the data are for reference only since they are obtained directly from the literature, where different references (total DNA/RNA, viral DNA/RNA, dilution fold, etc.) are used to analyze sensitivity.…”

“…Cao et al devised an RAA-LFT for the field detection of ToBRFV with high sensitivity, demonstrating a detection limit of 2.1 × 10 1 copies/50-µL reaction [39]. Subsequently, Zhao et al combined RAA and CRISPR/Cas12a with LFT, enabling the simultaneous detection of four tobamoviruses-pepper mild mottle virus (PMMoV), ToBRFV, tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV) [40].…”

“…This necessitates stringent requirements for primer design during the nucleic acid amplification process, a critical consideration not only for INAA-LFT but also for other nucleic-acid-based detection technologies. Furthermore, in the context of multiplex assays aiming to detect two or more distinct viruses, even within the same genus, the significance and complexity of the primer design are heightened-a formidable challenge for researchers [34,40].…”

“…In the currently established INAA-LFT detection system for plant viruses, researchers have diligently worked to consolidating multiple components into a single reaction tube (all in one) and simplify operational steps without compromising the efficacy of each component. Notably, processes such as reverse transcription are amalgamated into the amplification reaction, and nucleic acid amplification is seamlessly integrated with CRISPR/Cas12a within a single mixture [28,37,39,40]. Moreover, the development of a nucleic acid extraction-free process that is seamlessly incorporated into the all-in-one reaction further streamlines the INAA-LFT steps, enhancing simplicity [25,28,32,37,40].…”

“…Notably, processes such as reverse transcription are amalgamated into the amplification reaction, and nucleic acid amplification is seamlessly integrated with CRISPR/Cas12a within a single mixture [28,37,39,40]. Moreover, the development of a nucleic acid extraction-free process that is seamlessly incorporated into the all-in-one reaction further streamlines the INAA-LFT steps, enhancing simplicity [25,28,32,37,40].…”