Chemiluminescent detection of caprine arthritis encephalitis virus with a PCR-generated single stranded nonradiolabelled probe
| Autor: | Alfonso Clavijo, Jan Thorsen |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Male
Arthritis-Encephalitis Virus Caprine Molecular Sequence Data Dot blot Polymerase Chain Reaction Sensitivity and Specificity Microbiology Virus law.invention chemistry.chemical_compound law Animals Digoxigenin Caprine arthritis encephalitis virus Polymerase chain reaction Goat Diseases Base Sequence General Veterinary biology urogenital system Goats Hybridization probe Nucleic Acid Hybridization virus diseases General Medicine biology.organism_classification Virology Molecular biology chemistry Luminescent Measurements Lentivirus Infections Female Primer (molecular biology) DNA Probes Molecular probe |
| Zdroj: | Veterinary Microbiology. 43:295-305 |
| ISSN: | 0378-1135 |
| DOI: | 10.1016/0378-1135(94)00104-5 |
| Popis: | 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 slot 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. |
| Databáze: | OpenAIRE |
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