Reverse-transcription polymerase chain reaction for the detection of viruses from plants and aphids

A reverse transcription polymerase chain reaction (RT-PCR) protocol used for the detection of potato viruses in dormant tubers and leaves and in an aphid vector is described. Problems in plant sample preparation from different hosts, uneven distribution or low concentration of viruses and the presence of PCR inhibitors in plant extracts are discussed and various ways to eliminate their effect are described. Using Potyviridae viruses, it has been shown that RT-PCR in various modified forms can be used to differentiate viruses at the level of family, genus, species, strains and their subtypes or serotypes. The specificity of primer pairs and PCR modifications has been used to separate closely related potato viruses A and PVY strains (PVY(O), PVY(N) and PVY(NTN)) from a known mixture.