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. 1995 Feb;33(2):287–291. doi: 10.1128/jcm.33.2.287-291.1995

Rapid detection of bovine viral diarrhea virus by using RNA extracted directly from assorted specimens and a one-tube reverse transcription PCR assay.

A L Hamel 1, M D Wasylyshen 1, G P Nayar 1
PMCID: PMC227934  PMID: 7714180

Abstract

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.

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Selected References

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