Optimization of a simple, low-cost one-step reverse transcription recombinase polymerase amplification method for real-time detection of potato virus A in potato leaves and tubers

Vegetative propagation of potatoes makes it possible for potato viruses to be transmitted through tubers. Potato virus A (PVA) is one of these viruses, which belongs to the Potyvirus genus in the Potyviridae family. Potato tuber yield can be reduced by 30-40% by PVA alone. Losses can be further exacerbated by potato virus X and/or potato virus Y infection. PVA is transmitted primarily by several species of aphids in non-persistent manner. With the aim of resolving this problem, we developed one-step reverse transcription-recombinase polymerase amplification (RT-RPA), a highly sensitive and cost-effective method for detecting PVA in both potato tubers and leaves. Detection and amplification are performed using isothermal conditions in this method. There was good amplification of the coat protein gene in PVA with all three primers tested. To conduct this study, a primer set that can amplify specific 185 base pair (bp) product was selected. PVA detection was optimized by 30-min amplification reactions, which showed no cross-reactivity with other potato viruses. A simple heating block or water bath was used to amplify PVA product using RT-RPA at a temperature range of 38-42 C-degrees. In comparison to conventional reverse transcription-polymerase chain reaction (RT-PCR), the newly developed RT-RPA protocol exhibited high sensitivity for both potato leaves and tuber tissues. Using cellular paper-based simple RNA extraction procedure, the virus was detected in leaf samples as efficiently as purified total RNA. We also found that combining LiCl-based RNA precipitation with cellular paper discs allowed us to successfully optimize RNA extraction for one-step RT-RPA for detecting PVA in tubers. Tests using this simplified one-step RT-RPA method were successfully applied to 300 samples of both leaves and tubers from various potato cultivars. In our knowledge, this is the first report of an RT-RPA assay utilizing simple RNA obtained from either cellular disc paper or LiCl coupled with cellular disc paper to detect PVA. As a result, this method was equally sensitive and specific for detecting PVA in potatoes. The developed RT-RPA assay is more versatile, durable, and do not require highly purified RNA templates, thus providing an effective alternative to RT-PCR assays for screening of germplasm, certifying planting materials, breeding for virus resistance, and real-time monitoring of PVA.