CHEMILUMINESCENT DETECTION OF CAPRINE ARTHRITIS-ENCEPHALITIS VIRUS WITH A PCR-GENERATED SINGLE-STRANDED NONRADIOLABELLED PROBE

A 814-bp digoxigenin-labelled single stranded DNA probe was produced a nd utilized in slot-blot hybridization for detection of caprine arthri tis encephalitis virus (CAEV) in goat synovial membrane (GSM) cell cul ture infected with CAEV. The sensitivity of a PCR-generated probe was compared with a random primer labelled probe. The probe with digoxigen in-dUTP incorporated in the PCR reaction mixture was more sensitive fo r RNA detection than the random primer probe and it was much simpler t o use. The probe was applied for detection of CAEV by slot blot hybrid ization in peripheral blood mononuclear sells (PBMC) and macrophage cu ltures obtained from naturally infected goats. This technique was not sufficiently sensitive to detect the viral nucleic acid directly from PBMC or cultured macrophages. When macrophages were cultured in vitro and then cocultured with susceptible GSM cells, samples gave a positiv e signal in the slot-blot hybridization technique. The use of slot-blo t RNA hybridization permits more convenient and rapid confirmation of CAEV isolation in susceptible cells than the conventional identificati on by syncytia formation.