DETECTION OF AFRICAN HORSE SICKNESS VIRUSES BY DOT-BLOT HYBRIDIZATIONUSING A DIGOXIGENIN-LABELED PROBE

In order to develop a non-radioactive dot-blot hybridization assay, fo r the detection of African-horse sickness virus (AHSV), genome segment 7 from 9 serotypes was amplified by RT-PCR. The resulting PCR product s were denatured, immobilized on nylon membranes and then hybridized t o a non-radioactive digoxigenin-labelled probe. This probe (265 bp in length) was generated by nested-PCR using genome segment 7 of AHSV, se rotype 4 as a template. The dot-blot was visualized by chemiluminescen ce. Positives were obtained from the PCR products amplified from all 9 AHSV serotypes, but not from any other equine virus or orbivirus isol ates. The sensitivity and specificity of this probe, together with the simplicity and rapidity of this technique, suggest that a nonradioact ive dot blot assay may be useful as a method for the routine and rapid diagnosis of viral infections. (C) 1995 Academic Press Limited