Establishment of a real-time recombinase-aided isothermal amplification (RT-RAA) assay for the rapid detection of bovine respiratory syncytial virus (preprint)

ABSTRACT

Background Bovine respiratory syncytial virus (BRSV) is a major cause of bovine respiratory disease, resulting in significant losses to the cattle industry. For rapid detection of BRSV, reverse transcription recombinase polymerase amplification assays targeting the F gene were developed by integrating the fluorescence detection platform (RT-RAA).Results The developed RT-RAA assays allowed the exponential amplification of the target fragment in 20 min at a constant temperature of 39°C. The RT-RAA assays also showed good specificity for BRSV, with no cross-reactions with Infectious Bovine Rhinotracheitis Virus (IBRV), Bovine Parainfluenza Virus Type 3 (BPIV3), Bovine Viral Diarrhea Virus (BVDV) and Bovine Coronavirus (BCoV). With the standard RNA of BRSV serving as a template, the limit of detection for RT-RAA was 5 × 102 copies per reaction. Forty clinical samples collected from cattle with respiratory disease were tested, and the positive rate was 7.5% (3/40), consistent with results using the conventional PCR method reported previously.Conclusion An RT-RAA assay for BRSV detection was established in this study. The method is specific and sensitive and can be completed within 20 min at 39℃. These results ascertain that the developed RT-RAA assays are effective diagnostic tools for rapidly detecting BRSV in resource-limited settings, which may be applied for clinical detection of BRSV.