title: Nonradioactive, photobiotin-labelled DNA probes for routine diagnosis of viroids in plant extracts.
abstract: Avocado sunblotch viroid (ASBV), coconut cadang cadang viroid (CCCV), chrysanthemum stunt viroid (CSV) and potato spindle tuber viroid (PSTV) were detected in plant extracts by dot-blot hybridization using nonradioactive photobiotin-labelled nucleic acid probes. Recombinant DNA probes, containing full-length monomer viroid inserts in the plasmid vectors pSP64 or pUC9, were biotinylated with photobiotin and used as sonicated double-stranded DNA fragments. Using fresh leaf material, a general method (suitably modified for avocado tissue) was developed for the rapid preparation of purified nucleic acid extracts. Plant extracts from a range of field samples were spotted onto nitrocellulose, subjected to hybridization and the biotin-labelled DNA bound to the target nucleic acid was detected with an avidin-alkaline phosphatase conjugate. Under the stated hybridization and washing conditions, each individual viroid probe was specific. Each viroid was readily detected with a sensitivity similar to that obtained with the same (or a like) probe labelled with 32P. Healthy plant extracts gave colourless spots.
authors.affiliation: Department of Biochemistry, University of Adelaide, South Australia.
authors.forename: J L
authors.initials: JL
authors.lastname: McInnes
authors.forename: N
authors.initials: N
authors.lastname: Habili
authors.forename: R H
authors.initials: RH
authors.lastname: Symons
mesh_terms: D000345:Affinity Labels; D001386:Azides; D001710:Biotin / Q000031:analogs & derivatives*; D002021:Buffers; D003001:Cloning, Molecular; D003124:Colorimetry; D015342:DNA Probes; D005557:Formaldehyde; D015151:Immunoblotting / Q000379:methods; D010936:Plant Extracts / Q000032:analysis*; D012680:Sensitivity and Specificity; D014772:Viroids / Q000302:isolation & purification*
chemical_list: D000345:Affinity Labels; D001386:Azides; D002021:Buffers; D015342:DNA Probes; D010936:Plant Extracts; D005557:Formaldehyde; D001710:Biotin; C045549:photobiotin