Development of reverse transcription recombinase polymerase amplification assay for rapid diagnostics of Peanut mottle virus

Peanut mottle virus (PeMoV) is a single-stranded RNA virus transmitted through seeds and aphids that affects peanut crops worldwide. Currently, Enzyme Linked Immune-Sorbent assays and Reverse-Transcription Polymerase Chain Reaction techniques are widely employed to detect PeMoV in infected plants. ELISA is labor-intensive and time-consuming, as it involves the preparation of buffers and the production of polyclonal antibodies. Even though RT-PCR bypasses the need for buffer preparation and antibody production, it demands trained professional’s manpower, requires expensive equipment like thermal cyclers, and involves complex procedures such as RNA isolation and cDNA conversion. To avoid these constraints, there is a need for a fast, reliable, efficient, and economical method for detecting PeMoV to ensure the production of healthy seeds. This study optimized the Reverse Transcriptase Recombinase Polymerase Amplification (RT-RPA) method by eliminating the steps of RNA extraction, cDNA conversion, and the use of a thermal cycler. The optimized RT-RPA assay successfully detected PeMoV at concentrations as low as 10^–6 and 10^–7 dilutions (1 and 0.1 µg/µl) of both RNA an-6d crude sap templates, demonstrating high sensitivity comparable to the routine RT-PCR assay. The new RT-RPA technique was tested against other viruses that infect peanuts like the Peanut stunt Virus, Tomato spotted wilt virus and Peanut bud necrosis virus, this technique demonstrated great specificity and no cross-reactivity. The developed RT-RPA using a crude leaf sap template is time-saving, less laborious, not very complicated, high specificity, sensitivity, economical and efficient. Therefore, laboratories with limited resources can use the RT-RPA assay for preliminary screening of PeMoV in nurseries, farm and glasshouse conditions, and quarantine stations. The current study reports the development, optimization and validation of Reverse Transcriptase Recombinase Polymerase Amplification (RT-RPA) using crude sap as template for the onsite detection of PeMoV infection in peanut crops under field conditions for the first time.