A NOVEL NESTED REVERSE TRANSCRIPTION PCR DETECTS BOVINE VIRAL DIARRHEA VIRUS IN FLUIDS FROM ABORTED BOVINE FETUSES

A nested reverse transcription-PCR (RT-PCR) was developed to detect pe stivirus nucleic acid in fetal fluids and to study the number of bovin e abortions associated with BVDV infection. Three techniques for the e xtraction of viral RNA from fetal fluids were compared: phenol:chlorof orm method, treatment with Catrimox-14 followed by guanidium isothiocy anate buffer and the Qiagen total RNA kit. The Qiagen kit was the most sensitive and reproducible and therefore adopted. After cDNA synthesi s, initial amplification of a 288-base pair product using existing pri mers derived from the highly conserved 5'-untranslated region of the B VDV genome was achieved. Newly designed internal primers yielded a 171 -base pair fragment which was visualised after electrophoresis on an e thidium bromide-stained gel. This assay detected 6.0 TCID50 of BVDV pe r 300 mu l of artificially contaminated fetal fluid. One hundred fetal fluids were screened for the presence of BVDV RNA and the results com pared with existing virus isolation methods. The BVDV antibody status of each fetus was determined. The nested RT-PCR detected BVDV RNA in e ight of the hundred fetal fluids screened, whereas BVD virus was isola ted 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 BVD V. (C) 1998 Elsevier Science B.V. All rights reserved.