DETECTION OF BOVINE HERPESVIRUS-1 BY THE DOT-BLOT HYBRIDIZATION METHOD

With the development of molecular biology in the 1980s methods of micr oorganism detection start to innovate. One of the main adventages of t he molecular-genetic methods, namely hybridization of nucleic acids an d PCR methods, is the detection of genome of microorganism without the need for cellular cultivation. To detect BHV-1 (ethiological agens IB R-IPV) the dot-blot hybridization method on nitrocellulose filters was used together with different types of DNA probes (two-fiber recombina nt plasmids, one-fiber recombinant phages M 13 and 40 bp synthetic oli gonucleolid). Genome DNA BHV-1 was isolated from samples (virions, inf ested cells, nasal smears and secretions by phenol extraction). The hi ghest sensitivity of detection was achieved with P-32-pUR-1 probe (1.8 kb random EcoRI - Hind III fragment ligated into plasmid pUC 9) which detected genome BHV-1 in 5.10(3) infested MDBK cells. This probe did n ot respond with herpetic viruses BHV-2, BHV-3, BHV-4 and the virus of Aujeszky's disease. The quality of pUR-1 probe was further tested for IBR diagnostics in animals experimentally infested with the virus BHV- 1 (intranasal infection). BHV-1 could be detected in nasal smears and secretions in experimentally infested calves as early as on the first day following infection, while the agens amount reached its peak on th e days 2-3 and on the days 6-7 the occurrence of virus fell markedly. When digoxigenin-pUR-1, i.e. non-radioactively marked probe, the virus presence was confirmed only on the days 2-3, in the time of the highe st occurrence of infection agens. To detect the virus through the dot- blot hybridization nasal secretions were confirmed as better compared with nasal smears. The technology of virus isolation on cell cultures confirmed also the occurrence of agens as soon as on the first day fro m infection, with maximum on the days 2-5, but much more reliably it d etected the virus on the days observed from the day 3 and their peak w as obtained on the day 6 from infection. Experiments, comparing classi cal methods of IBR diagnostics (detection of specific antibodies, the method of isolation on cellular cultures) with the dot-blot hybridizat ion using the samples obtained from farms with natural occurrence of I BM, are under progress.