Rapid identification of potato virus Y strains by one-step triplex RT-PCR.
A one-step triplex RT-PCR method was characterised that allows rapid, strain-specific detection of potato virus Y (PVY) occurring on potato: PVY(N), PVY(O), PVY(NTN) (recombinant isolates), PVY(N)Wi and PVY(C). Three specific primer pairs were designed on aligned PVY sequences available from genomic data banks. The specificity of the selected primers was first examined by simplex RT-PCR with a large number of PVY reference isolates. Two fragments of 0.44 and 1.11kb were amplified for PVY(N) and non-recombinant PVY(NTN) isolates, two fragments of 0.53 and 0.66kb for PVY(O) isolates, a single fragment of 0.44kb for recombinant PVY(NTN) isolates, a 0.66kb fragment for PVY(C) isolates and a 0.53kb fragment for PVY(N)Wi isolate. The primers were then combined in a one-step triplex RT-PCR reaction, optimised stepwise and validated with the reference isolates. The great similarity between the genomes of PVY(N) and non-recombinant PVY(NTN) prevented their differentiation using this method. No fragments were amplified with samples infected by non-related potato viruses, as well as with samples from healthy tobacco and potato plants. The one-step triplex RT-PCR described here fastens specific detection of PVY strains that are otherwise only distinguishable by combined serological and biological assays.
Rigotti
Stefania
S
Agroscope Changins-Wädenswil Research Station ACW, Department of Virology, CH-1260 Nyon 1, Switzerland.
Gugerli
Paul
P
D017931:DNA Primers; D016679:Genome, Viral; D021141:Nucleic Acid Amplification Techniques; D010935:Plant Diseases / Q000821:virology*; D017800:Potyvirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000821:virology*; D013997:Time Factors; D014026:Nicotiana / Q000821:virology*
D017931:DNA Primers; D012367:RNA, Viral