A new approach for the simultaneous differentiation of biological and geographical strains of Potato virus Y by uniplex and multiplex RT-PCR

Abstract

Two strains of Potato virus Y (PVY), the common (PVY O) and the tobacco veinal necrosis (PVY N) have been known for decades. More recently, a tuber ringspot necrosis (PVY NTN), and several recombinants of PVY O and PVY N (designated here as PVY N:O) have been described. Further, the PVY N group of strains have been assigned to two geographical subgroups of European (EU) PVY N/NTN and the North American (NA) PVY N/NTN. The evolution of new PVY N strains, has complicated the diagnosis, which requires a combination of bioassay, serological and molecular assays. To simplify the identification and differentiation of various PVY N strain groups, a competitive (single antisense and multiple sense primers) reverse transcription-polymerase chain reaction (RT-PCR) was used, making use of minor differences in the variable region part of the PVY genome. Specifically, primers based on small variations in nucleotide stretches of P1 gene permitted a broad range separation of PVY O and PVY N groups and the specific detection of strain subgroups. The primer pairs designed for identifying PVY O, EU-PVY N/NTN, NA-PVY N and NA-PVY NTN are described. Primer pairs can be used in a uniplex (single pair of primer) or multiplex (duplex, tetraplex or pentaplex) competitive RT-PCR, allowing simultaneous testing for any combination of PVY O, EU-PVY N/NTN, NA-PVY N and NA-PVY NTN.