Quantitative detection of cucumber mosaic cucumovirus RNA with microplate hybridization using digoxigenin-labeled oligo-deoxyribonucleotide probe

A simple microplate hybridization method was developed for subgroup-specifi c and quantitative detection of RNA of cucumber mosaic cucumovims (CMV). Di goxigenin (DIG) -labeled, synthetic oligo-deoxyribonucleotides were used as the probes for hybridization. CMV RNAs were adsorbed to microplate wells a nd then hybridized with the probes specific for the respective subgroups of CMV. Under optimal conditions, about one ng of CMV RNA was detected withou t any nonspecific reactions. When total RNAs extracted from tobacco plants infected with CMV-Y, CMV-m2 or mock-inoculated were subjected to the hybrid ization, we found no cross-reactions between viral RNAs of subgroup I and I I and no nonspecific reactions against healthy controls. The microplate hyb ridization method had several advantages over conventional dot blot hybridi zation methods.