Chemiluminescent detection of potato and pome fruit viroids by digoxigenin-labeled dot blot and tissue blot hybridization

Abstract

A chemiluminescent molecular hybridization protocol was compared to 32P autoradiography for detecting potato spindle tuber viroid (PSTVd) and apple scar skin group viroids (ASSVd). Labeled cRNA probes for PSTVd and ASSVd were synthesized by SP6 RNA polymerase transcription using digoxigenin-11-UTP or alpha-[32P]UTP. Dot blot hybridization of purified viroids and sap extracts from infected plants showed that chemiluminescent detection using digoxigenin-labeled probes was as sensitive as autoradiography using 32P probes. A minimum of 2.0-2.5 pg purified viroid was detected. ASSVd could be detected in as little as 0.4 ng of total nucleic acid extract from infected tissue or in sap extracts diluted to 10(-3) with healthy extracts. Tissue blots of PSTVd-infected potato tubers and tomato roots, stems and leaves and ASSVd-infected apple fruit, stems and petioles, gave positive reactions when hybridized with the digoxigenin probe. No reaction with similar tissues from healthy plants was observed.