A simple detection method for potato viruses combining template preparation by heat treatment of homogenate with primers and one-step multiplex RT-PCR

Potatoes are susceptible to viral diseases, and widespread viral infections lead to reduced production yields. Potato leaf roll virus, potato virus S, potato virus X, and potato virus Y are important viruses predominant in potato fields and cause significant economic damage in Japan. In this study, to reduce the labor and cost of virus testing in seed certification program, a simple molecular diagnostic assay for these potato viruses was developed. This method consists of template preparation by heat-treating a mixture of leaf homogenate and a newly designed primer set, followed by one-step conventional multiplex RT-PCR (mRT-PCR). In particular, to simplify RNA extraction, a procedure was adopted in which leaf homogenates in PBST were diluted 100-fold in RNase-free water, heat-treated with primers for 5 min at 70°C and subjected to one-step mRT-PCR. Interestingly, heat treatment of a mixture of the homogenate and primers improved the detection sensitivity for the virus. This new method’s detection sensitivity of each virus was 10–100 times higher than that of ELISA using commercial kits and 1–10 times higher than that of one-step mRT-PCR with filter paper-based RNA extraction. The heat-treated homogenate with primers was also applicable for detecting eight other potato viruses by one-step conventional mRT-PCR and four potato viruses by real-time mRT-PCR which our research group previously developed. New direct RT-PCR method for potato viruses can be applied to seed certification programs, where a large number of samples need to be tested.