Use of Primers with 5' Non-complementary Sequences in RT-PCR for the Detection of Nepovirus Subgroups A and B

Two generic PCR protocols were developed to detect nepoviruses in subgroups A and B using degenerate primers designed to amplify part of the RNA-dependent RNA polymerase (RdRp) gene. It was observed that detection sensitivity and specificity could be improved by adding a 12-bp non-complementary sequence to the 5' termini of the forward, but not the reverse, primers. The optimized PCR protocols amplified a specific product ( approximately 340bp and approximately 250bp with subgroups A and B, respectively) from all 17 isolates of the 5 virus species in subgroup A and 3 species in subgroup B tested. The primers detect conserved protein motifs in the RdRp gene and it is anticipated that they have the potential to detect unreported or uncharacterised nepoviruses in subgroups A and B.