Development of a New Gene Diagnosis Method for Plant Viruses and Viroids

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1 : To setablish nucleic acid hybridization assay protocol that is carried in mult-well microplates, we have examined several factors affecting the sensitivity. Nucleic acids (mainly RNA) were dfficiently adsorbed on polystirene multi-well microplate by denaturing in a solution containing 50% formamide, 6.125% formaldehyde, 1X SSC,for 10 min at 68C.Hybridization procedure was greatly simplified comparing to the traditional membrane hybridization by eliminating several steps that are essential for membrane hybridization, that is, a part of washing, RNase A treatment and pre-hybridization steps.Finally by using digoxigenine (DIG)-labeled cRNA and p-nitrophenylphosphate for the probe and substrate, we have established a simple and sensitive microplate hybridization (MPH) that is sensitive enough to detect picograms of virus and viroid RNAs.2 : We have further examined the sensitivity of the MPH method by comparing to the traditional serological diagnosis method, such as ELISA.In case of hop latent viruse, MPH was 40 to 100 times more sensitive that ELISA.3 : We have developed MPH probes specific for ten viroids (HSVd, CEVd, ASSVd, PBCVd, PSTVd, HLVd, CbVd1, GYSVd, CSVd and CVdIII) and eight viruses (CMV,CMV-satellite RNA,HLV,TuMV,WCMV,MNSV,PVY and HMV).