Chemiluminescent detection of Fiji disease virus with biotinylated DNA probes.
Biotinylated Fiji disease fijivirus specific cDNA probes detected the presence of the virus in total nucleic acid extracts from infected sugarcane plants. Hybridised biotinylated probes were detected with streptavidin-alkaline phosphatase conjugate and the light generating substrate AMPPD. Samples were either blotted manually, or by alkaline capillary transfer using 100 mM NaOH. Transfer of nucleic acids to charge modified nylon with sodium hydroxide was superior to denaturation with glyoxal or formamide and salt-citrate buffer transfer as the bands were clearly resolved and no degradation of the FDV dsRNA was observed. Transfer of either total nucleic acid extracts or purified dsRNA in manifold blots generated false positive signals with the non-radio-active chemiluminescent detection systems tested. Manual or northern blots had a limit of detection for purified target double-stranded RNA of approximately 10 pg and 0.5 pg respectively. Manual blots were tested for practical application to screen germplasma for FDV infection. The virus was detected in leaf samples from FDV-infected plants, in some instances prior to development of the characteristic gall symptom.
David North Plant Research Centre, Bureau of Sugar Experiment Stations, Indooroopilly, Queensland, Australia.
G R
GR
Smith
M L
ML
Clarke
R
R
Van de Velde
J L
JL
Dale
D001710:Biotin; D015152:Blotting, Northern; D015342:DNA Probes; D008163:Luminescent Measurements; D010944:Plants / Q000382:microbiology*; D012330:RNA, Double-Stranded / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis; D012087:Reoviridae / Q000235:genetics / Q000302:isolation & purification*
D015342:DNA Probes; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D001710:Biotin