Digoxigenin-labelled cDNA probes for the detection of potato virus Y in dormant potato tubers.
An isolate (Y139) of the potato virus Y strain PVYO, common in the potato-growing regions of eastern Canada, was cloned and segments of about 1800 bases from the 3'-end of the PVY-genome were sequenced. These clones were used to prepare molecular detection probes by labelling with digoxigenin. Five methods reported to yield nucleic acid preparations from tuber tissue were compared. Two of the methods failed to yield nucleic acids from tubers suitable for use in dot-blot hybridization. The other three methods yielded satisfactory nucleic acids which could be successfully detected with the PVYO probes. A method of nucleic acid extraction from tuber tissues was selected which is simple, reproducible and does not use phenol. Using this standardized procedure, PVY was detected in freshly harvested, greenhouse-grown tubers of 8 potato cultivars; the probe was specific to PVY; and the sensitivity of detection using purified viral RNA was down to 10 pg. The sensitivity of detection in tuber extracts was down to a dilution of 1:64, while that from potato leaves was to a dilution of 1:1024. Nucleic acids prepared by the standardized procedure and denatured prior to storage at 4 degrees C, -20 degrees C or at -70 degrees C remained suitable for hybridization for over 2 weeks.
Singh
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Agriculture and Agri-Food Canada, Fredericton Research Centre, New Brunswick.
Singh
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D015342:DNA Probes; D018076:DNA, Complementary; D004279:DNA, Viral / Q000032:analysis; D004076:Digoxigenin; D007202:Indicators and Reagents; D017800:Potyvirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000821:virology*
D015342:DNA Probes; D018076:DNA, Complementary; D004279:DNA, Viral; D007202:Indicators and Reagents; D004076:Digoxigenin