IDENTIFYING BOVINE RESPIRATORY SYNCYTIAL VIRUS BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION AND OLIGONUCLEOTIDE HYBRIDIZATIONS

An assay to identify tissue culture cells infected with bovine respira tory syncytial virus (BRSV) that utilizes reverse transcription (RT), the polymerase chain reaction (PCR), and a synthetic oligonucleotide h ybridization probe has been developed. The RT-PCR assay uses a BRSV-sp ecific negative-sense oligonucleotide primer to synthesize cDNA from a BRSV fusion protein mRNA template and another BRSV-specific oligonucl eotide primer (positive sense) upstream from the negative-sense primer for PCR amplification. In the presence of mRNA templates of BRSV isol ates originating from locations throughout the United States, the BRSV RT-PCR assay resulted in amplified products (381 bp) that were specif ic to BRSV, as demonstrated in hybridizations with a positive-sense ol igonucleotide probe complementary to internal sequences and in sequenc e comparisons with the F protein of BRSV 391-2. In analysis of the BRS V 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 produc t with a BRSV-specific oligonucleotide probe will greatly add to the s peed, sensitivity, and specificity of BRSV diagnostics.