Durán-Vila, Núria

Palacio-Bielsa, Ana

ReDivia - Repositorio Digital de l'Instit Valencià d'Investigacions Agràries

IMPRINT HYBRIDIZATION FOR DETECTION OF CITRUS VIROIDS.A. Palacio € N. Duran-VilaDepartamento de Protección Vegetal y Biotecnología. Instituto Valenciano deInvestigaciones Agrarias. Moncada (Valencia). Spain.Commercial citrus are vulnerable to viroid induced diseases, exocortis and cachexia-xyloporosis. Availability of fast and reliable indexing methods 1s critical to implementsanitation, quarantine and certification programs, and to identify trees suspected of beinginfected with viroids.In the past citrus viroids were detected by biological indexing using Etrog citron 861-S1 and Parson's Special mandarin as indicators. However, it was demonstrated that nucleicacid analysis of inoculated citrons by sequential gel electrophoresis (SPAGE) provided afaster and more reliable method for the routine indexing ofall citrus viroids. This methodwhich combines the desirable characteristics of citron as an excellent host for viroids and thesensitivity of nucleic acid analysis was adopted at IVIA for viroid indexing.Recently, an imprint hybridization method has been designed to simplify theprocessing of citron samples. Freshly cut stem tissues from inoculated citrons are imprintedby firmly press the cut surface onto positively charged Nylon membranes. The imprintedsamples once fixed on the membranes, can be processed immediately or stored. Processing ofthe imprinted membranes involves: a) preparation of DIG labeled probes (RNA probessynthesized by in vitro transcription or DNA probes synthesized by PCR using cloned DNArelated to the specific viroid sequences); b) hybridization of the membranes against the DIG-labeled viroid probes; c) detection of DIG-labeled hybrids (using anti-DIG-alkalinephosphatase conjugate); d) visualization of the DIG-alkaline phosphatase (using thechemiluminiscence substrate CSPD). Probes for the five viroid groups identified in citrushave been assayed and shown to be highly specific. In addition a single hybridization using amixture of the five probes allows a fast screening for viroid-free versus viroid containingsamples. The overall process is simple and can be easily performed by non-specializedpersonnel including some nurserymen. Membranes containing positive and negative controlsand the chemicals needed (including the mixtures of labeled probes) could be easilycommercialized as detection kits.Efforts are now being devoted to adapt this imprmnt hybridization method for thedetection of citrus viroids directly from commercial varieties.

Imprint Hybridation  For Detection Of Citrus Viroids

http://redivia.gva.es/bitstream/20.500.11939/6599/1/1997_Palacio_Imprint.pdf

Imprint Hybridation For Detection Of Citrus Viroids

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