Rapid detection of bovine viral diarrhea virus by using RNA extracted directly from assorted specimens and a one-tube reverse transcription PCR assay.
We describe a simple method for the rapid detection of bovine viral diarrhea virus (BVDV) that uses a one-tube reverse transcription PCR (RT-PCR) and total RNA extracted directly from a variety of bovine specimens, including whole blood and tissues. Reagents for both RT and PCR were combined in a one-tube, single-buffer system, and amplification was performed with a single uninterrupted thermal cycling program. Using the novel cationic surfactant tetradecyltrimethylammonium oxalate (Catrimox-14), we consistently extracted RT-PCR-quality RNA from specimens containing blood. Amplification with primers derived from conserved sequences within the BVDV 5'-untranslated region yielded a 244-bp product. Assay specificity was confirmed by ethidium bromide-stained gel electrophoresis and by chemiluminescence-assayed Southern blot hybridizations involving BVDV 5'-untranslated region-specific digoxigenin-labelled cDNA probes. The assay detection level was 0.1 50% tissue culture infectious dose of BVDV when ethidium bromide-stained gel electrophoresis was used and 0.01 50% tissue culture infectious dose of BVDV when Southern blot hybridization was used. Our method is an alternative to the conventional cell culture assays used in a diagnostic laboratory and is an improvement over existing RT-PCR assays for BVDV.
Hamel
A L
AL
Virology Laboratory, Manitoba Agriculture Veterinary Services, Winnipeg, Canada.
Wasylyshen
M D
MD
Nayar
G P
GP
D000818:Animals; D001483:Base Sequence; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis* / Q000517:prevention & control / Q000821:virology; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D003902:Detergents; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics* / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D000644:Quaternary Ammonium Compounds; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D050337:Trimethyl Ammonium Compounds; D014773:Virology / Q000379:methods
D017931:DNA Primers; D004279:DNA, Viral; D003902:Detergents; D000644:Quaternary Ammonium Compounds; D012367:RNA, Viral; D050337:Trimethyl Ammonium Compounds; C009410:tetradecyltrimethylammonium