Rapid identification of potato virus Y strains by one-step triplex RT-PCR

Abstract

A one-step triplex RT-PCR method was characterised that allows rapid, strain-specific detection of potato virus Y (PVY) occurring on potato: PVY N, PVY O, PVY NTN (recombinant isolates), PVY NWi and PVY C. Three specific primer pairs were designed on aligned PVY sequences available from genomic data banks. The specificity of the selected primers was first examined by simplex RT-PCR with a large number of PVY reference isolates. Two fragments of 0.44 and 1.11 kb were amplified for PVY N and non-recombinant PVY NTN isolates, two fragments of 0.53 and 0.66 kb for PVY O isolates, a single fragment of 0.44 kb for recombinant PVY NTN isolates, a 0.66 kb fragment for PVY C isolates and a 0.53 kb fragment for PVY NWi isolate. The primers were then combined in a one-step triplex RT-PCR reaction, optimised stepwise and validated with the reference isolates. The great similarity between the genomes of PVY N and non-recombinant PVY NTN prevented their differentiation using this method. No fragments were amplified with samples infected by non-related potato viruses, as well as with samples from healthy tobacco and potato plants. The one-step triplex RT-PCR described here fastens specific detection of PVY strains that are otherwise only distinguishable by combined serological and biological assays.