Detection of African horse sickness viruses by dot-blot hybridization using a digoxigenin-labelled probe.
In order to develop a non-radioactive dot-blot hybridization assay, for the detection of African-horse sickness virus (AHSV), genome segment 7 from 9 serotypes was amplified by RT-PCR. The resulting PCR products were denatured, immobilized on nylon membranes and then hybridized to a non-radioactive digoxigenin-labelled probe. This probe (265 bp in length) was generated by nested-PCR using genome segment 7 of AHSV, serotype 4 as a template. The dot-blot was visualized by chemiluminescence. Positives were obtained from the PCR products amplified from all 9 AHSV serotypes, but not from any other equine virus or orbivirus isolates. The sensitivity and specificity of this probe, together with the simplicity and rapidity of this technique, suggest that a non-radioactive dot blot assay may be useful as a method for the routine and rapid diagnosis of viral infections.
D001483:Base Sequence; D004076:Digoxigenin; D015151:Immunoblotting / Q000379:methods*; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D018163:Orbivirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012330:RNA, Double-Stranded / Q000032:analysis*
D012330:RNA, Double-Stranded; D004076:Digoxigenin