Abstract: Several test conditions for the simultaneous detection of potato leafroll (PLRV) and potato virus Y (PVY) in dormant tubers and leaves by reverse transcription - polymerase chain reaction (RT-PCR) were optimized. Various factors optimized at the reverse transcription (RT) stage rather than at the amplification (PCR) stage affected the outcome. Two-fold dNTP's concentration (1.0mM) is required during RT to yield distinct bands of PLRV and PVY in duplex PCR in comparison to 1- fold dNTP's (0.5mM) for the simplex PCR. Various proportions of antisense primers of PLRV and PVY used during RT affected subsequent duplex RT-PCR. Optimal amplification of both viruses were obtained at a ratio of 0.90:0.49uM of PLRV:PVY antisense primers. Similarly, in the multiplex PCR oligo dT:PLRV antisense primer (1.0uM:0.65uM) at RT stage amplified distinctly PVY, PLRV, PVS/PVM viruses with a primer combination of oligo dT 1.0uM, PVY sense primer 1.0uM, Carla uni 0.4uM and PLRV antisense and sense primer 0.4um at the PCR stage. An interaction of dNTP's and RNA template concentration was observed. A higher concentration of RNA was required at the one-fold dNTP's concentration than at 2-fold dNTP's. Application of optimized conditions to singly- and doubly- infected tubers detected both PLRV and PVY from naturally infected field grown tubers. Similarly, multiplex RT-PCR detected PVY, PLRV, PVM/PVS from naturally infected plants, but its application to dormant tubers need extensive lab and field evaluation.
1Agricultural Certification Services Inc. / N.B. Potato Agency, Comp. 17, 385 Wilsey Road, Fredericton, NB E3B 5N6