Detection of persistent bovine viral diarrhea virus infections by DNA hybridization and polymerase chain reaction assay

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Abstract
The detection of persistent bovine viral diarrhea virus (BVDV) infections in cattle by DNA dot blot hybridization was done with a cloned cDNA probe prepared from noncytopathic BVDV (strain NY-1). Due to the variability of specific hybridization results, detection of BVDV by primer-directed polymerase chain amplification was done. Primers were chosen within a reported area of sequence conservation and amino acid homology of Pestiviruses. The amphfication region extended from nucleotide 6322 to 7475 and was based on published BVDV sequence data (NADL strain). BVDV RNA was extracted by two methods (proteinase K and guanidinium isothiocyanate) from serum and white blood cell preparations collected from 3 persistently-infected heifers. cDNA was synthesized from extracted BVDV-genomic RNA using reverse transcriptase. Reaction conditions were optimized to amplify the 1153 base pair fragment from the cDNA preparation. Detection of BVDV in the samples by DNA dot blot hybridization using a nucleic acid probe corresponding to the amplified region (6322 to 7475) was compared with polymerase chain reaction assay. The increased sensitivity of the polymerase chain reaction assay provided clearer identification of persistently-infected animals than DNA hybridization under similar conditions.