Submitted to: American Phytopathological Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 8/1/2001
Publication Date: N/A
Technical Abstract: A rapid and sensitive assay for the specific detection of plant viroids using RT-PCR-probe capture hybridization (RT-PCR-ELISA) was developed. Using each viroid specific DNA primers and capture probes the assay was applied successfully for the detection of the following viroids from their respective infected tissues: potato spindle tuber, chrysanthemum stunt, avocado sunblotch, peach latent mosaic, and apple scar skin. Clarified sap extract from infected leaf tissue was treated first with GeneReleaser (TM) polymeric matrix to remove inhibitors of RT-PCR reactions. Viroid cDNA was then synthesized and amplified using viroid specific primers in RT-PCR assays and the amplified viroid cDNA (amplicon) was DIG-labeled during the amplification process using a modified one tube RT-PCR-DIG labeled method. The amplicon was then detected in a colorimetric hybridization system in a microtiter plate using a biotinylated cDNA or cRNA capture probe. This system combines the specificity of molecular hybridization, the ease of the colorimetric protocol, and is at least 100 fold more sensitive than gel electrophoretic analysis in detecting the amplified product. Viroid cRNA may replace viroid cDNA as the capture probe. Six to seven hours are needed to complete the RT-PCR-ELISA assay for viroid detection from infected leaf tissue.