Establishment of a one-step reverse transcription recombinase polymerase amplification assay for the detection of...

Potato virus S (PVS) is a noteworthy threat to the propagation of healthy seed potatoes. Accurate and speedy detection is critical for effective PVS management. In the present study, an isothermal-based one-step reverse transcription-recombinase polymerase amplification (RT-RPA) approach was developed to detect PVS infection in potato leaves and tubers. A primer set based on the coat protein gene successfully amplified a 158 bp product out of three primer sets examined. The amplification reaction took less than 30 min to complete with no account of cross-reactivity with major potato viruses. Additionally, amplification of RT-RPA products was performed on the heating system and/or water bath at 38-42 °C. The results of sensitivity analysis revealed that one-step RT-RPA has shown 100 times higher sensitivity than routine RT-PCR for the detection of PVS in infected leaves. Furthermore, ten times higher sensitivity of RT-RPA was observed in infected tubers. The methodology was simplified further by the use of template RNA extracted using a cellular disc paper-based extraction method that detected the PVS more effectively than purified total RNA. PVS was detected in 175 samples (leaves and tubers each) of several potato varieties using this innovative technique. To our acquaintance, this is the first report of one-step RT-RPA using a basic RNA extract derived through cellular disc paper that is significantly sensitive and precise for PVS detection in potatoes. The advantages of one-step RT-RPA in terms of proficiency, robustness, and the availability of a highly pure RNA template make it an attractive choice for seed accreditation, resistance breeding, and field inspections.