Single and Double Polymerase Chain Reaction for Detection of Bovine Viral Diarrhea Virus in Tissue Culture and Sera

Abstract

Bovine viral diarrhea virus (BVDV) is an ubiquitous pathogen of cattle and has been reported in other ruminants. It is also frequently present in laboratory and biological materials as an adventitious agent. This virus is difficult to detect in some specimens, especially in the presence of specific antibody and when the virus is present in low concentrations. In this paper, we describe a single polymerase chain reaction (PCR) to amplify virus sequences from infected cell culture and a nested double PCR to detect small concentrations of several virus strains in sera. Total cellular RNA was extracted from cell cultures infected with the cytopathic strain 72 and noncytopathic strain 2724 of BVDV. Ten different genomic sequences along the length of the viral RNA ranging in size from 397 to 1,016 base pairs (bp) were successfully amplified by PCR. A 404-bp probe made from amplified product from the 3′ end hybridized specifically with the RNA of several BVDV strains blotted on nylon filters. Viral RNA was extracted from serum and amplified using 2 sets of degenerate nested primers designed from the 3′ end of the viral genome in a double PCR protocol. Double amplification of the viral sequences greatly enhanced the sensitivity of the detection of many strains present in serum. Advantages of using double PCR over single PCR and virus isolation is discussed.