Summary
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 germplasm 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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Anon (1986) Instructions for use of PR800 hybridisation chamber. Hoeffer Scientific Instruments, San Franciso
Anon (1989a) Bionick labelling system protocol. BRL, Gaithersburg,
Anon (1989b) ECL technical update. Amersham International, England
Asamizu T, Summers D, Motika MB, Anzula JV, Nuss DL (1985) Molecular cloning and characterization of the genome of wound tumor virus: a tumor-inducing plant reovirus. Virology 144: 398–409
Dale JL, Phillips DA, Parry JN (1986) Double-stranded RNA in banana plants with bunchy top disease. J Gen Virol 67: 371–375
Davis VS, Boyle JA (1990) Adapting the polymerase chain reaction to a double stranded RNA genome. Anal Biochem 189: 30–34
Gubler V, Hoffman BI (1983) A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269
Holmes IH (1991) Reoviridae. In: Franck RIB, Fauquet CM, Knudson DL, Brown F (eds) Classification and Nomenclature of Viruses. Fifth Report of the International Committee on Taxonomy of Viruses. Springer, Wien New York, pp 186–199 (Arch Virol [Suppl] 2)
Kowalik TA, Yang Y-Y, Li JK-K (1990) Molecular cloning and comparative sequence analyses of bluetongue virus S1 segments by selective synthesis of specific full-length DNA copies of dsRNA genes. Virology 177: 820–823
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbour Laboratory, New York
Parker B, Li JK-K (1988) A method to produce six alkaline northern blots of viral dsRNA within one hour. Biotechniques 6: 22–23
Parker B, Li JK-K (1988) Alkaline northern blotting of dsRNA to Zeta-probe membranes. Mol Biol Rep 4: 4–5
Reddy DVR, Boccardo G, Outridge R, Teakle DS, Black LM (1975) Electrophoretic separation of dsRNA genome segments from Fiji disease and maize rough dwarf viruses. Virology 63: 287–291
Skotnicki AH, Dale JL, Skotnicki ML (1986) Detection of Fiji disease virus in infected sugarcane by nucleic acid hybridisation. J Virol Methods 13: 71–77
Smith GR, Van de Velde R, Dale JL (1992) PCR amplification of a specific double-stranded RNA region of Fiji disease virus from diseased sugarcane. J Virol Methods 39: 237–246
Upcroft P, Healy A (1987) Rapid and efficient method for cloning blunt-ended DNA fragments. Gene 51: 69–75
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Smith, G.R., Clarke, M.L., Van de Velde, R. et al. Chemiluminescent detection of Fiji disease virus with biotinylated DNA probes. Archives of Virology 136, 325–334 (1994). https://doi.org/10.1007/BF01321061
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01321061