A. Clavijo et J. Thorsen, CHEMILUMINESCENT DETECTION OF CAPRINE ARTHRITIS-ENCEPHALITIS VIRUS WITH A PCR-GENERATED SINGLE-STRANDED NONRADIOLABELLED PROBE, Veterinary microbiology, 43(4), 1995, pp. 295-305
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.