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, treatmen...

Full description

Saved in:
Bibliographic Details
Published in:Journal of virological methods 1998-03, Vol.71 (1), p.69-76
Main Authors: Hyndman, L, Vilcek, S, Conner, J, Nettleton, P.F
Format: Article
Language:English
Subjects:
ABORTION
Abortion, Veterinary - virology
ABORTO
ACIDE NUCLEIQUE
ACIDOS NUCLEICOS
Amniotic Fluid - virology
Animals
Antibodies, Viral - blood
ARN
AVORTEMENT
Biological and medical sciences
BOVINAE
Bovine Virus Diarrhea-Mucosal Disease - virology
Cattle
DIARREA
Diarrhea Viruses, Bovine Viral - genetics
Diarrhea Viruses, Bovine Viral - isolation & purification
DIARRHEE
DIARRHOEA
Female
Fetal fluids
FETO
Fetus - virology
FOETUS
Fundamental and applied biological sciences. Psychology
Microbiology
NUCLEIC ACIDS
PESTIVIRUS
Polymerase Chain Reaction - methods
Polymerase Chain Reaction - veterinary
Pregnancy
Reagent Kits, Diagnostic
Reproducibility of Results
Reverse transcription-polymerase chain reaction
RNA
RNA extraction
RNA, Viral - genetics
Sensitivity and Specificity
Techniques used in virology
Virology
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 TCID 50 of BVDV per 300 μl 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.
ISSN:0166-0934
1879-0984
DOI:10.1016/S0166-0934(97)00206-1