Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/5/1999
Publication Date: N/A
Interpretive Summary: Viroids such as potato spindle tuber, peach latent mosaic, and apple scar skin cause significant crop damage, hamper crop production, and are of quarantine importance to the U.S. and abroad. Rapid and specific detection of these viroids is prerequisite for their management and control. Detection of these pathogens by reverse transcription - polymerase chain reaction (RT-PCR) technology has become commonplace. Amplified PCR products are usually detected by electrophoresis on agarose or polyacrylamide gels. After electrophoresis, gels must be stained with ethidium bromide or silver nitrate in order to visualize the results. Silver nitrate staining often results in a high background and the staining solution is a hazardous waste. Ethidium bromide is less sensitive than silver nitrate, is a carcinogen, and must be specially treated before disposal. To facilitate and expedite detection, we developed a simplified, rapid procedure for the analysis of viroid-specific RT-PCR products. This method employs the format of an enzyme-linked immunosorbent assay (ELISA) and liquid phase nucleic acid hybridization. The method is safer, faster, more specific and sensitive, more adaptable to large scale automation, and the results are more easily visualized. These qualities make the developed method (RT-PCR-ELISA) an attractive tool for diagnosis of plant pathogens. This method should greatly assist in detecting and controlling viroids through certification and quarantine programs world-wide.
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. The assay was applied successfully for the detection of potato spindle tuber viroid, peach latent mosaic viroid, or apple scar skin viroid from viroid infected leaf tissue. 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. The amplicon was then detected in a colorimetric hybridization system in a microtiter plate using a biotinylated cDNA 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.