Use of polymerase chain reaction to simultaneously detect and type bovine viral diarrhoea viruses isolated from clinical specimens.

The techniques of indirect immunofluorescence (IF), immuno-peroxidase (IP) staining and the one-step reverse transcriptase polymerase chain reaction (RT-PCR) were compared for detection of 102 isolates of bovine viral diarrhoea virus (BVDV) in infected cell cultures. The BVDV was obtained from bovine clinical specimens, including sera, buffy coats and tissues, submitted from farms located in the States of Iowa and Wisconsin, United States of America. The IF technique detected 88/102 (86.3%) of the viral isolates, whereas IP staining detected an additional 4 isolates (92/102; 90%). The one-step RT-PCR using primers derived from the 5' untranslated region of the BVDV genome detected 102/102 (100%) of the BVDV isolates. A second-round PCR utilising another pair of PCR primers from the 5' untranslated region, allowed rapid genotyping of BVDV. The procedure used showed that the PCR assay based on the 5' untranslated region of the virus genome is the most sensitive indicator for BVDV detection in cell culture, and is also of considerable epidemiological importance since it allowed rapid genotyping of BVDV isolated from clinical specimens. In addition to detection and genotyping of BVDV isolated from clinical specimens, the RT-PCR procedure can be used for routine screening of locally produced and imported biologicals for BVDV contamination. However, the procedure requires further refinement to enable direct application on the clinical specimen.