Detection of four major potato viruses in Japan using a simple RNA preparation and one-step multiplex RT-PCR

A high-throughput alternative to ELISA to detect potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY), economically important viruses in Japan, has been needed for seed potato production. To develop an alternative using a multiplex reverse transcription polymerase chain reaction (mRT-PCR), we verified reported primers by two-step mRT-PCR and designed new primers for PVS and PVY based on the conserved region of genome sequences among three lineages of PVS or six strains of PVY. In addition, primers specific for potato and tobacco elongation factor 1 alpha were designed as an internal control for mRT-PCR. With these primers, one-step mRT-PCR detected all reference isolates of the four viruses. A published paper-based RNA preparation was modified for use in a single tube with an elution procedure to preserve RNA as evidence. As a rinse solution to wash away contaminants, 50% (v/v) isopropanol and 75% (v/v) ethanol resulted in a sensitivity for all viruses was tenfold higher than with the buffered detergent. In the mRT-PCR, the optimized paper-based RNA preparation gave tenfold higher sensitivity than the other two RNA preparation methods. Compared with ELISA, the new RNA preparation with the mRT-PCR was tenfold more sensitive for PVY and PLRV, 1,000-fold more for PVX, but the same for PVS. The new detection method simultaneously detected the four potato viruses from potato leaf by group test and is efficient and sensitive enough to detect the four potato viruses for seed potato certification, quarantine, breeding, and field surveys.