Rapid diagnosis of rabies infection by means of a dot hybridization assay

Abstract

Dot hybridization was used to detect specific rabies RNA in brains, either from experimental infection in mouse or from brain material to be processed for routine diagnosis. 32P cDNA probes were employed to identify minute amounts of specific viral RNA. Purified RNA was obtained after phenol extraction. The RNA was fixed on nylon membranes and hybridized with a pool of M13 inserts complementary to 200-400 nucleotides of each rabies gene and mRNA. Hybridized, labelled probes were detected by autoradiography. There was strong cross-hybridization between fixed rabies and street rabies virus RNA, which enable the detection of field strains for diagnosis purpose using a fixed rabies (PV strain) cDNA. A positive response was obtained with as little as 80 ng of brain RNA material from a fixed rabies-infected mouse. Detection of viral RNA was still specific 1 week after death, the brain material being left at room temperature. A total correlation was found when the samples were examined in parallel using a fluorescent rabies-specific antibody and by virus isolation on murine neuroblastoma cells. These data show that the use of rabies-specific cDNA probes in a dot-blot hybridization assay has great potential for the diagnosis of rabies.