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

One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y

“…All viral isolates in the previous report were also used in this study [ 13 ]. The viral isolates were propagated in mechanically inoculated potato plant cv.…”

“…The viral isolates were propagated in mechanically inoculated potato plant cv. Irish Cobbler (IC) and virus infection was confirmed by conventional RT-PCR [ 13 ]. Virus-infected leaves were stored at − 80 °C until use.…”

“…PCR Procedure was as follows. The RNA solution was extracted using a simple filter paper-based method [ 13 ]. The RNA solution was stored at − 80 °C until use, and 1 µl of the supernatant was used as the template.…”

“…Toward meeting this need, multiplex reverse transcription-polymerase chain reaction (mRT-PCR)-based assays are becoming common [ 9 – 12 ]. We have already developed one-step conventional mRT-PCR assays, combined with a filter-paper-based simple RNA preparation for the four major potato viruses and a plant gene as internal control [ 13 ]. The advantage of this protocol is that as cost-effective as the ELISA it yields results more rapidly; however, the subject is still time-consuming for the electrophoretic analysis and the risk of PCR products contamination by open-tube manipulation.…”

“…Here, we developed a new one-step real-time mRT-PCR assay for the four major potato viruses and used a plant gene as the internal control. Combined with the simple RNA preparation method [ 13 ], the detection of Japanese isolates was demonstrated. The reliability of calculating Tm values were confirmed by comparing measured and calculated Tm values for Japanese representatives.…”