Nonradioactive, photobiotin-labelled DNA probes for routine diagnosis of viroids in plant extracts
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Date
1989
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Elsevier
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 photo-biotin-labelied nucleic acid probes. Recombinant DNA probes, containing full-length monomer viroid inserts in the plasmid vectors pSP64 or pUC9, were bio-tinylated 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 hybrid- ization 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.
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Citation
Journal of Virological Methods,23(1989) 299-312