一种用于检测枸橼中柑橘裂皮类病毒的快速灵敏斑点杂交测定法，采用地高辛标记的RNA探针。

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

作者信息

Fonseca M E, Marcellino L H, Gander E

机构信息

Laboratorio de Biologia Molecular, Centro Nacional de Pesquisa de Recursos Geneticos e Biotecnologia (CENARGEN), EMBRAPA, Brazil.

出版信息

J Virol Methods. 1996 Apr 5;57(2):203-7. doi: 10.1016/0166-0934(95)01976-6.

DOI:10.1016/0166-0934(95)01976-6

PMID:8801232

Abstract

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

摘要

为了检测感染柑橘的粗汁液中的柑橘裂皮类病毒（CEVd），开发了一种使用体外转录的地高辛标记RNA探针（核糖探针）的快速灵敏斑点杂交检测方法。该方案包括使用甲醛变性步骤从样品中非常快速简单地制备RNA提取物。根据我们的结果，强烈建议采用这一步骤，因为与未经过甲醛处理的提取物相比，甲醛变性样品中的杂交信号明显更强。该检测方法灵敏度足以检测0.1 ng纯化的CEVd RNA，并且能够在0.2 mg有症状的柑橘叶片中检测到类病毒。核糖探针的使用还允许在高温条件下进行杂交，避免非特异性背景。