Podleckis E V, Hammond R W, Hurtt S S, Hadidi A
United States Department of Agriculture, National Germplasm Resources Laboratory, Beltsville, MD 20705-2350.
J Virol Methods. 1993 Jul;43(2):147-58. doi: 10.1016/0166-0934(93)90072-y.
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.
将一种化学发光分子杂交方法与32P放射自显影法进行比较，以检测马铃薯纺锤块茎类病毒（PSTVd）和苹果锈果类病毒组（ASSVd）。使用地高辛-11-UTP或α-[32P]UTP通过SP6 RNA聚合酶转录合成用于PSTVd和ASSVd的标记cRNA探针。对纯化的类病毒和来自受感染植物的汁液提取物进行斑点印迹杂交表明，使用地高辛标记探针的化学发光检测与使用32P探针的放射自显影一样灵敏。检测到的纯化类病毒最少为2.0 - 2.5 pg。在感染组织的总核酸提取物低至0.4 ng或与健康提取物稀释至10(-3)的汁液提取物中均可检测到ASSVd。用PSTVd感染的马铃薯块茎以及番茄的根、茎和叶，以及用ASSVd感染的苹果果实、茎和叶柄进行组织印迹，与地高辛探针杂交时呈阳性反应。未观察到与健康植物的类似组织发生反应。
