Zastosowanie real time RT-PCR do wykrywania wirusa biegunki bydła i choroby błon śluzowych (BVD-MD) typu 1 i typu 3 w materiale biologicznym

The aim of the study was to use real time RT-PCR for the detection of genetic material of bovine viral diarrhea virus (BVDV) type 1 and type 3 in serum and milk samples. Material tested included the fetal calf serum used for cell culture, serum samples from healthy calves and from calves experimentally inoculated with BVDV type 1 and type 3, and milk samples (pasteurized and treated with ultra high temperature). Sensitivity of the test was 200 copies of RNA per reaction (10⁵ viral RNA copies per ml) for both types of BVDV, using dedicated primers and standards. Detection limit was 10² tissue culture infectious dose 50 (TCID₅₀) and 1 TCID₅₀ for type 1 and type 3, respectively. Diagnostic specificity of the method was 100%. Out of 10 samples of milk, 3 were positive for BVDV type 1, while none was positive for type 3. On the other hand, BVDV type 3 was found in 6 out of 10 samples of fetal calf serum. Real Time RT-PCR for BVDV type 1 and type 3 proved to be a highly sensitive and highly specific technique, enabling the detection of viral genetic material in various samples, even when its detection by virus isolation or antigen ELISA tests is impossible because of virus inactivation by such processes as high temperature, gamma irradiation, or the presence of virus neutralizing antibodies.