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. 2024 Mar 27:11:1360504.
doi: 10.3389/fvets.2024.1360504. eCollection 2024.

Establishment and application of a rapid diagnostic method for BVDV and IBRV using recombinase polymerase amplification-lateral flow device

Yan Wang  1 , Jinyuan Shang  1 , Zhijie Li  1 , Ao Zhang  1 , Yuening Cheng  1
Affiliations

Establishment and application of a rapid diagnostic method for BVDV and IBRV using recombinase polymerase amplification-lateral flow device

Yan Wang et al. Front Vet Sci. .

Abstract

Bovine Viral Diarrhea Virus (BVDV) and Infectious Bovine Rhinotracheitis Virus (IBRV) are the two most prevalent infectious diseases in cattle. They both can cause persistent infection and immunosuppression, resulting in significant economic losses in the livestock industry. Therefore, rapid detection of early BVDV and IBRV infections is crucial. In this study, a method for the rapid detection of BVDV and IBRV was established by using recombinase polymerase amplification (RPA) combined with lateral flow device (LFD). By optimizing the temperature and time conditions of the RPA reaction, the sensitivity, specificity, and clinical performance were evaluated. The results indicated that the RPA reaction could be completed at 40°C within 25 min. The LOD for BVDV and IBRV by RPA-LFD were 5.1 × 101 copies/μL and 6.65 × 101 copies/μL, respectively, with no cross-reactivity observed with other viruses such as CSFV, BRSV, BPIV3, BRV, and BCoV. Testing of 32 clinical samples showed consistent results between RPA-LFD and qPCR. The RPA-LFD method established in this study can be used for the rapid clinical detection of BVDV and IBRV, which providing a rapid and convenient molecular biology approach for on-site rapid detection and epidemiological investigations. Simultaneously, it offers technical support for the prevention and control of these viruses.

Keywords: Bovine Viral Diarrhea Virus; Infectious Bovine Rhinotracheitis Virus; lateral flow dipstick; rapid detection; reverse transcriptase recombinase aided amplification.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of LFD.
Figure 2
Figure 2
Optimizing detection conditions in the RPA-LFD assay. (A) The reaction temperature of BVDV-RPA ranged from 25°C to 50 °C. (B) The reaction time of BVDV-RPA ranged from 5 min to 30 min.
Figure 3
Figure 3
Optimizing detection conditions in the RPA-LFD assay. (A) The reaction temperature of IBRV-RPA ranged from 25 °C to 50 °C. (B) The reaction time of IBRV-RPA ranged from 5 min to 30 min.
Figure 4
Figure 4
Analyzing the duplex RPA-LFD method sensitivity. (A) The dual RPA-LFD detection system can detect 51.0 copies/reaction standards BVDV plasmids. (B) The LOD of the method was 66.5 copies IBRV plasmids per reaction. (C) The sensitivities and standard curve of RealStar Green Fast Mixture real-time PCR established for the 10-fold serial dilutions of BVDV and IBRV standard RNA. Lanes 1–7: 107, 106, 105, 104, 103, 102, and 101 copies of the template, 8: Mock.
Figure 5
Figure 5
Analyzing the duplex RPA-LFD method specificity. Lanes 1 to 8: BVDV and IBRV, IBRV, BVDV, CSFV, BRSV, BPIV3, BRV, and BCoV; Lane 8: Mock.
Figure 6
Figure 6
RPA-LFD detection results for 32 clinical samples. Results of qPCR detection for 32 clinical samples of BVDV and IRBV.
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