A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.
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.
Fonseca
M E
ME
Laboratorio de Biologia Molecular, Centro Nacional de Pesquisa de Recursos Geneticos e Biotecnologia (CENARGEN), EMBRAPA, Brazil.
Marcellino
L H
LH
Gander
E
E
D004076:Digoxigenin; D005069:Evaluation Studies as Topic; D005557:Formaldehyde; D005638:Fruit / Q000821:virology; D009693:Nucleic Acid Hybridization; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D015347:RNA Probes; D012367:RNA, Viral / Q000032:analysis / Q000187:drug effects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014772:Viroids / Q000235:genetics / Q000302:isolation & purification*
D015347:RNA Probes; D012367:RNA, Viral; D005557:Formaldehyde; D004076:Digoxigenin