Improvement in the sensitivity of PVY^N detection by increasing the cDNA probe size

Cloned cDNA to a North American isolate of tobacco veinal necrotic strain of potato virus Y (PVY^N) RNA was used for the detection of PVY^N in diseased samples. Digoxigenin-labelled cDNA probes were prepared from ∼ 0.56 kb, ∼1.2 kb, ∼ 2.5 kb, and ∼ 3.25 kb overlapping clones representing the 3′-terminus of the PVY^N 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 PVY^N was maximum using the largest probe and the sensitivity generally decreased with a decrease in probe size. Five pg of homologous purified PVY^N RNA was detected with the ~ 3.25 kb probe, and 1000 pg of PVY^N RNA was required for the ~ 0.56 kb probe. The different levels of sensitivity of detection were also apparent when crude nucleic acid extracts from PVY^N and PVY^O infected plants were used. In comparison with tobacco bioassay, and dot immunobinding assay nucleic acid hybridization was found to be more sensitive.