Identifying bovine respiratory syncytial virus by reverse transcription-polymerase chain reaction and oligonucleotide hybridizations

Abstract

An assay to identify tissue culture cells infected with bovine respiratory syncytial virus (BRSV) that utilizes reverse transcription (RT), the polymerase chain reaction (PCR), and a synthetic oligonucleotide hybridization probe has been developed. The RT-PCR assay uses a BRSV-specific negative-sense oligonucleotide primer to synthesize cDNA from a BRSV fusion protein mRNA template and another BRSV-specific oligonucleotide primer (positive sense) upstream from the negative-sense primer for PCR amplification. In the presence of mRNA templates of BRSV isolates originating from locations throughout the United States, the BRSV RT-PCR assay resulted in amplified products (381 bp) that were specific to BRSV, as demonstrated in hybridizations with a positive-sense oligonucleotide probe complementary to internal sequences and in sequence comparisons with the F protein of BRSV 391-2. In analysis of the BRSV RT-PCR assay with prototype strains of human RSV subgroups A and B, amplification of a similar 381-bp RT-PCR product was not evident, and no RT-PCR product hybridized with the internal probe. We conclude that the specific ability to amplify DNA sequences of BRSV F protein mRNA by RT-PCR and then to demonstrate the presence of the amplified product with a BRSV-specific oligonucleotide probe will greatly add to the speed, sensitivity, and specificity of BRSV diagnostics.