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

Two strains of Potato virus Y (PVY), the common (PVYO) and the tobacco veinal necrosis (PVYN) have been known for decades. More recently, a tuber ringspot necrosis (PVYN:N), and several recombinants of PVYO and PVYN (designated here as PVYN:O) have been described. Further, the PVYN group of strains have been assigned to two geographical subgroups of European (EU) PVYN/NTN and the North American (NA) PVYN/NTN. The evolution of new PVYN strains, has complicated the diagnosis, which requires a combination of bioassay, serological and molecular assays. To simplify the identification and differentiation of various PVYN 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 PI gene permitted a broad range separation of PVYO and PVYN groups and the NA-PVYN specific detection of strain subgroups. The primer pairs designed for identifying PVYO, EU-PVYN/NTN, NA-PVYN and NA-PVYNTN 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 PVYO, EU-PVYN/NTN, NA-PVYN and NA-PVYNTN. (C) 2002 Elsevier Science B.V. All rights reserved.