Sensitive detection of potato spindle tuber and temperate fruit tree viroids by reverse transcription-polymerase chain reaction-probe capture hybridization

A rapid and sensitive assay for the specific detection of plant viroids usi ng reverse transcription-polymerase chain reaction (RT-PCR) -probe capture hybridization (RT-PCR-enzyme-linked immunosorbent assay (ELISA)) was develo ped. The assay was applied successfully for the detection of potato spindle tuber viroid, peach latent mosaic viroid, or apple scar skin viroid from v iroid infected leaf tissue. Clarified sap extract from infected leaf tissue was treated first with GeneReleaser(TM) polymeric matrix to remove inhibit ors of RT-PCR reactions. Viroid cDNA was then synthesized and amplified usi ng viroid specific primers in RT-PCR assays and the amplified viroid cDNA ( amplicon) was digoxigenin (DIG) -labelled 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 comb ines the specificity of molecular hybridization, the ease of the colorimetr ic protocol, and is at least 100-fold more sensitive than gel electrophoret ic analysis in detecting the amplified product. Viroid cRNA may replace vir oid cDNA as the capture probe. The cRNA probe was several fold more sensiti ve than the cDNA probe for viroid detection. Six to seven hours are needed to complete the RT-PCR-ELISA for viroid detection from infected leaf tissue . Published by Elsevier Science B.V.