# Developing a recombinase-aided amplification method combined with a lateral flow dipstick assay for rapid triplex detection of bovine coronavirus, infectious bovine rhinotracheitis virus, and bovine viral diarrhea virus

## TL;DR

This study created a fast and accurate test to detect three cattle viruses at once, making it easier to diagnose and manage diseases in cows.

## Contribution

The development of a triplex RAA-LFD assay for simultaneous detection of BCoV, IBRV, and BVDV in cattle.

## Key findings

- The triplex RAA-LFD assay detected all three viruses within 20 minutes at 39°C with high sensitivity and specificity.
- The method showed strong agreement with RT-qPCR (kappa coefficients 0.88–0.94) in clinical validation with 73 bovine samples.
- The assay had no cross-reactivity with other bovine viruses and detection thresholds of 10³ copies/μL for each virus.

## Abstract

To establish a sensitive, simple rapid method for detecting bovine coronavirus (BCoV), infectious bovine rhinotracheitis virus (IBRV), and bovine viral diarrhea virus (BVDV). Recombinase-aided amplification (RAA) was used to amplify template DNA or cDNA, and a lateral flow dipstick (LFD) was used to interpret the results after amplification was completed. Seventy-three rectal and nasal bovine samples were tested to evaluate the performance of the RAA-LFD assay and were tested in parallel via polymerase chain reaction (PCR) and reverse transcription quantitative PCR (RT-qPCR) for comparison. The triplex RAA-LFD assay was completed within 20 min at 39°C. This method demonstrated reasonable specificity, with no cross-reactivity to bovine norovirus (BNoV), bovine rotavirus (BRV), bovine parainfluenza virus 3 (BPIV3), or bovine respiratory syncytial virus (BRSV). The detection thresholds for IBRV, BVDV, and BCoV are 3.20 × 103 copies/μL, 2.21 × 103 copies/μL, and 4.13 × 103 copies/μL, respectively. Triplex RAA-LFD practicality was tested on 73 rectal and nasal bovine swabs from diarrheic cattle on commercial farms. Compared with RT-qPCR, the triplex RAA-LFD assay yielded a clinical sensitivity of 98.41%, 80.00%, and 85.71%, a positive predictive value (PPV) of 100.00%, and kappa coefficients of 0.94, 0.88, and 0.91 for BCoV, IBRV, and BVDV, respectively. Compared with PCR, it achieved 100.00%, 75.00%, and 87.50% sensitivity; 41.94%, 75.00%, and 58.33% PPV; and 0.33, 0.73, and 0.65 kappa coefficients for BCoV, IBRV, and BVDV, respectively. A triplex RAA-LFD method was developed to simultaneously detect BCoV, IBRV, and BVDV, offering an efficient solution for identifying multiple cattle viral pathogens.

This study developed a triplex RAA-LFD assay for the simultaneous detection of BCoV, IBRV, and BVDV. The method demonstrated high sensitivity (detection limits: 10³ copies/μL), specificity (no cross-reactivity with related viruses), and speed (20 min, including amplification and visualization). Clinical validation with 73 bovine samples showed strong agreement with RT-qPCR (kappa coefficients: 0.88–0.94), supporting its reliability. Compared with that of PCR, the sensitivity of this method remains high although the PPV varies. This rapid, low-cost, field-deployable assay improves surveillance of co-infections in cattle, aiding timely diagnosis and disease management, particularly in resource-limited settings.