Specific differentiation of recombinant PVYN:O and PVYNTN isolates by multiplex RT-PCR

The recombinant isolates of tobacco veinal necrotic strain of Potato virus Y (PVYN) and potato tuber necrotic group (PVYNTN) contain segments of the PVYO and the PVYN genome. Three major recombinant junctions (RJ) are present in the genome of the recombinant PVTNTN at sites HC/Pro-P3, 6K2-NIa, and the C-terminal region of CP gene and one RJ at HC/Pro-P3 site in some recombinant PVYN isolates (termed PVYN:O). Protocols for specific differentiation of the recombinant PVYNTN and PVYN:O from the non-recombinant PVYN are described. Specific primer pairs were designed to target the three RJs so that sense and antisense primers completely matched the nucleotide sequences at either side of the RJ. In a uniplex reverse transcription-polymerase chain reaction (RT-PCR), the first primer pair amplified a fragment of 641 bp from the recombinant PVYNTN and PVYN:O. The second and third primer pairs exclusively amplified fragments of 448 and 290 bp, respectively from the recombinant PVYNTN. In a multiplex (triplex) RT-PCR, when all three primer pairs were used simultaneously, the three fragments (641, 448 and 290 bp) were amplified exclusively from the recombinant PVYNTN, while only one fragment (641 bp) was amplified from the PVYN:O isolates, clearly differentiating the two recombinant isolates. No amplification was observed from the non-recombinant PVY, including PVYO and North American (NA)-PVYN/NTN. For further improvement of the multiplex RT-PCR, effects of cDNA preparation using specific antisense primers, random primers or oligo(dT) plus random primers were investigated. The cDNA prepared by random primer plus oligo(dT) increased the overall band intensity. (C) 2003 Elsevier B.V. All rights reserved.