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 and utilized in slot-blot hybridization for detection of caprine arthritis encephalitis virus (CAEV) in goat synovial membrane (GSM) cell culture infected with CAEV. The sensitivity of a PCR-generated probe was compared with a random primer labelled probe. The probe with digoxigenin-dUTP incorporated in the PCR reaction mixture was more sensitive for RNA detection than the random primer probe and it was much simpler to use. The probe was applied for detection of CAEV by blot blot hybridization in peripheral blood mononuclear cells (PBMC) and macrophage cultures 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 positive signal in the slot-blot hybridization technique. The use of slot-blot RNA hybridization permits more convenient and rapid confirmation of CAEV isolation in susceptible cells than the conventional identification by syncytia formation.
Clavijo
A
A
Department of Veterinary, Microbiology and Immunology, University of Guelph, Ontario, Canada.
Thorsen
J
J
D000818:Animals; D016088:Arthritis-Encephalitis Virus, Caprine / Q000302:isolation & purification*; D001483:Base Sequence; D015342:DNA Probes / Q000096:biosynthesis*; D005260:Female; D015511:Goat Diseases / Q000821:virology*; D006041:Goats; D016180:Lentivirus Infections / Q000662:veterinary* / Q000821:virology; D008163:Luminescent Measurements; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity
D015342:DNA Probes