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 (∼340 bp and ∼250 bp 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.