IMPROVEMENT IN THE SENSITIVITY OF PVYN DETECTION BY INCREASING THE CDNA PROBE SIZE

Cloned cDNA to a North American isolate of tobacco veinal necrotic str ain of potato virus Y (PVYN) RNA was used for the detection of PVYN in diseased samples. Digoxigenin-labelled cDNA probes were prepared from similar to 0.56 kb, similar to 1.2 kb, similar to 2.5 kb, and similar to 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 templa te cDNA used. The sensitivity of detection of PVYN was maximum using t he largest probe and the sensitivity generally decreased with a decrea se in probe size. Five pg of homologous purified PVYN RNA was detected with the similar to 3.25 kb probe, and 1000 pg of PVYN RNA was requir ed for the similar to 0.56 kb probe. The different levels of sensitivi ty of detection were also apparent when crude nucleic acid extracts fr om 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.