Improvement in the sensitivity of PVYN detection by increasing the cDNA probe size.
Cloned cDNA to a North American isolate of tobacco veinal necrotic strain of potato virus Y (PVYN) RNA was used for the detection of PVYN in diseased samples. Digoxigenin-labelled cDNA probes were prepared from approximately 0.56 kb, approximately 1.2 kb, approximately 2.5 kb, and approximately 3.25 kb overlapping clones representing the 3'-terminus of the PVYN genome. It was shown that the probe sizes, as determined by alkaline gel-electrophoresis, generally corresponded to the size of the template cDNA used. The sensitivity of detection of PVYN was maximum using the largest probe and the sensitivity generally decreased with a decrease in probe size. Five pg of homologous purified PVYN RNA was detected with the approximately 3.25 kb probe, and 1000 pg of PVYN RNA was required for the approximately 0.56 kb probe. The different levels of sensitivity of detection were also apparent when crude nucleic acid extracts from PVYN and PVYO infected plants were used. In comparison with tobacco bioassay, and dot immunobinding assay nucleic acid hybridization was found to be more sensitive.
Dhar
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Agriculture and Agri-Food Canada, Fredericton Research Centre, Canada.
Singh
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RP
D015342:DNA Probes; D018076:DNA, Complementary / Q000032:analysis; D015151:Immunoblotting; D009693:Nucleic Acid Hybridization; D017800:Potyvirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity
D015342:DNA Probes; D018076:DNA, Complementary; D012367:RNA, Viral