Comparison of nonradioactive cDNA probes for detection of potato spindle tuber viroid by dot-blot hybridization assay.
Six nonradioactive cDNA probes were compared for their sensitivities for detecting potato spindle tuber viroid (PSTVd) by dot-blot hybridization assay. Three biotinylated PSTVd cDNA probes, labeled by photoactivation with photobiotin, by nick translation or by random priming with biotinylated deoxyribonucleotides, were all capable of detecting 20 pg of purified PSTVd by a colorimetric assay and 2-20 pg by a chemiluminescent assay. Digoxigenin-labeled probe was able to detect 200 pg of purified PSTVd. Two biotinylated probes prepared with polymerase chain reaction (PCR) incorporating biotinylated dUTP or dATP were the most sensitive: 0.2-2 pg of PSTVd was detectable by both assays. All six probes could detect PSTVd also in extracts of infected tomato leaves at a dilution of up to 1/250-1/1250. These nonradioactive probes are equal to radioactive probes in their sensitivity, and the biotinylated probes produced with PCR amplification are particularly suitable for practical diagnosis, as they are sensitive and rapidly prepared in large quantities.
Kanematsu
S
S
Faculty of Agriculture, University of Tokyo, Japan.
Hibi
T
T
Hashimoto
J
J
Tsuchizaki
T
T
D001483:Base Sequence; D001710:Biotin / Q000031:analogs & derivatives; D003124:Colorimetry; D015342:DNA Probes; D015151:Immunoblotting; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000302:isolation & purification*; D010944:Plants / Q000382:microbiology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014772:Viroids / Q000302:isolation & purification*
D015342:DNA Probes; D001710:Biotin