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.