Rapid and simple detection of alfalfa mosaic virus using RT-RPA-CRISPR-Cas12a based lateral flow assay in alfalfa crops in China

Abstract

Alfalfa mosaic virus (AMV) has been reported as one of the most common and serious alfalfa-infecting viruses, significantly impacting alfalfa production worldwide. To establish a rapid and simple method for AMV detection in alfalfa crops in China, three novel methods were developed, including real-time fluorescent reverse-transcription recombinase polymerase amplification (RT-RPA), a one-tube one-step RT-RPA-CRISPR-Cas12a-based fluorescent assay, and a one-tube one-step RT-RPA-CRISPR-Cas12a-based lateral flow assay. RT-RPA-CRISPR-Cas12a-based detection methods combine one-step RT-RPA with CRISPR-Cas12a-based fluorescent or lateral flow detection in a single tube. Considering sensitivity, reaction time, ease of operation, and portability of instruments and reagents, the one-tube one-step RT-RPA-CRISPR-Cas12-based lateral flow assay was identified as the most suitable method for rapid field detection of AMV. This assay utilizes a pair of specific RT-RPA primers and a specific crRNA designed based on the conserved multifunctional coat protein gene sequences from 26 AMV isolates. The assay could be completed in approximately 1 h under isothermal conditions at 42 °C and 37 °C using a portable metal incubator. Sensitivity tests demonstrated that the assay could detect as low as 9.0 pg of total RNA extracted from AMV-infected alfalfa plants collected from Inner Mongolia, Hebei, Beijing, and Shanxi provinces in China, with no cross-reactivity with other alfalfa-infecting viral pathogens. Furthermore, its feasibility was validated by testing field-collected alfalfa samples. In conclusion, the developed one-tube one-step RT-RPA-CRISPR-Cas12a-based lateral flow assay offers a simple, sensitive, and efficient method for rapid AMV detection in the field, showing significant potential for practical applications.