Nonradioactive, photobiotin-labelled DNA probes for routine diagnosis of viroids in plant extracts.
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.
McInnes
J L
JL
Department of Biochemistry, University of Adelaide, South Australia.
Habili
N
N
Symons
R H
RH
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*
D000345:Affinity Labels; D001386:Azides; D002021:Buffers; D015342:DNA Probes; D010936:Plant Extracts; D005557:Formaldehyde; D001710:Biotin; C045549:photobiotin