The feasibility of tetraplex RT-PCR in the determination of PVS, PLRV, PVX and PVY from dormant potato tubers

Abstract

Dormant potato tubers belonging to cvs., namely, Agria, Granola and Marfona known to be infected with potato viruses (

Potato leafroll virus, PLRV; Potato virus S, PVS; Potato virus X, PVX and Potato virus Y, PVY) were tested with uniplex RT-PCR and strong bands specific to each virus were obtained from cultivars. When cDNA synthesized for uniplex RT-PCR was used for tetraplex RT-PCR, the bands obtained from PVS, PLRV and PVX were too faint to be photographed and there is no any observed band for PVY. To improve the band density, the concentration of oligo dT primer in RT was increased from 20 to 40 ng in the subsequent experiments. The increasing of oligo dT primer concentration in RT increased the band density for PVS and PVX, but not PVY. Upon this, different amount of total RNA were tested in RT stage. The best result was obtained from 5 μl of total RNA and followed by 3.5 and 2.5 μl applications. In order to determine the effect of cDNA amount in PCR, 2 μl cDNA + 23 μl PCR, 5 μl cDNA + 20 μl PCR and 5 μl cDNA + 25 μl PCR mixture were compared. However, no distinct differences were observed among various cDNA amounts. As a result, instead of tetraplex RT-PCR, it is suggested the use of triplex RT-PCR for reliable detection of PLRV, PVS and PVX. However, uniplex PCR could be suggested for reliable detection of PVY from this study by using the same cDNA.