A novel nested reverse transcription PCR detects bovine viral diarrhoea virus in fluids from aborted bovine fetuses.

A nested reverse transcription-PCR (RT-PCR) was developed to detect pestivirus nucleic acid in fetal fluids and to study the number of bovine abortions associated with BVDV infection. Three techniques for the extraction of viral RNA from fetal fluids were compared; phenol:chloroform method, treatment with Catrimox-14 followed by guanidium isothiocyanate buffer and the Qiagen total RNA kit. The Qiagen kit was the most sensitive and reproducible and therefore adopted. After cDNA synthesis, initial amplification of a 288-base pair product using existing primers derived from the highly conserved 5'-untranslated region of the BVDV genome was achieved. Newly designed internal primers yielded a 171-base pair fragment which was visualised after electrophoresis on an ethidium bromide-stained gel. This assay detected 6.0 TCID50 of BVDV per 300 microl of artificially contaminated fetal fluid. One hundred fetal fluids were screened for the presence of BVDV RNA and the results compared with existing virus isolation methods. The BVDV antibody status of each fetus was determined. The nested RT-PCR detected BVDV RNA in eight of the hundred fetal fluids screened, whereas BVD virus was isolated from only one sample. The use of the nested RT-PCR will provide us with a more accurate picture of bovine embryonic infection due to BVDV.