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.
Wei
Ting
T
Plant Health and Environment Laboratory, Investigation and Diagnostic Centre, MAF Biosecurity New Zealand, P.O. Box 2095, Auckland 1140, New Zealand.
Clover
Gerard
G
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D017860:Nepovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000821:virology; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase