Factors affecting detection of PVY in dormant tubers by reverse transcription polymerase chain reaction and nucleic acid spot hybridization.
A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two 20-mer primers located in nuclear inclusion genes NIa and NIb of potato virus Y (PVY). A 1017 bp PCR-product was detected in dormant potato tubers, infected with PVY(O), but not in tubers from healthy plants. The PCR product was specific to PVY, as determined by Southern blot detection by hybridization with a PVY(O)-specific probe. As little as 1 pg of purified PVY(O)-RNA can be detected after RT-PCR amplification. The presence of phenolics or polysaccharides in tuber nucleic acids inhibited PVY(O) amplification, which was eliminated by diluting nucleic acid preparations prior to cDNA synthesis, modifying the nucleic acid extraction procedure by isopropanol precipitation and using phosphate-buffered saline-Tween in the cDNA mix. Potato cultivars differed in PVY(O) concentration in tubers as much as 128-fold. Tuber parts used for nucleic acid extractions were important in potato cultivars with low virus titres and did not result in reduced detection of PVY(O) by both nucleic acid spot hybridization and RT-PCR, but RT-PCR band intensity was lower at longer storage periods. The primer pair developed in this study exhibited broad specificities with field isolates from Peru, Scotland and North America.
Singh
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Agriculture and Agri-Food Canada, Fredericton Research Centre, New Brunswick, Canada.
Singh
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RP
D004279:DNA, Viral / Q000032:analysis; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D017800:Potyvirus / Q000235:genetics / Q000302:isolation & purification*; D014158:Transcription, Genetic
D004279:DNA, Viral