Rapid visual detection of bovine viral diarrhea virus (BVDV) using recombinase polymerase amplification with SYBR Green I

Abstract

Abstract Bovine diarrhea virus (BVDV) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, with clinical manifestations of fever, diarrhea, ulcers, and abortions, which cause significant economic losses to the cattle industry. The establishment of an efficient, rapid and sensitive assay suitable for field conditions is conducive to the early detection of pathogens and the implementation of relevant treatments. Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification method that has been widely used in the diagnosis of infectious diseases. In this paper, an RPAS assay for recombinase polymerase amplification combined with SYBR Green I was developed for the rapid detection of BVDV. The assay was completed at a constant temperature of 37℃ for 25 min, and the minimum detection limit of RPA was 1×101 copies/µL for gel electrophoresis. Under sunlight, the minimum detection limit of BVDV RPAS visualization was 1×109 copies/µL; Under UV, the minimum detection limit of BVDV RPAS was 1×105 copies/µL. The assay has no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory tract viruses. Clinical samples had equal BVDV RPA, RPAS, and PCR detection thresholds, and SYBR Green I visualization was evident. In conclusion, the BVDV-RPAS established in this study, with high sensitivity and specificity, has the potential to be used as a powerful tool for BVD prevention and control.