Objective A rapid and visualizable method for detecting bovine parainfluenza virus type 3 (BPIV3) was developed utilizing the reverse transcription-recombinase polymerase amplification (RT-RPA), magnetic capture, and microfluidic chips.

Methods Specific primers and probes were designed based on the BPIV3 gp3 gene. The bovine viral diarrhea virus (BVDV), bovine adenovirus type 3 (BADV3), and bovine infectious rhinotracheitis virus (IBRV) were used as control to determine assay specificity. Assay sensitivity was examined on a 10x dilution of RNA extraction solution of the standard BPIV3 strain. Fifty-six serum and nasal swab samples from the cattle suspected of acute BPIV3 infection were collected for detection using the newly established method. Test results were compared with those obtained on nasal swab samples by RT-PCR.

Results The amplicon from the RNA extract of the standard BPIV3 strain sized 404bp, with none detected in control, obtained by the assay. A 100% homology with the highly conserved gp3 gene NC_002161.1 of BPIV3 was shown. At different concentrations of the extract, the lowest assay detection limit was 2.26×10³ copies·L⁻¹. From the 56 serum samples, the cows, coded SD0078, SD0114, SD0319, SD0601, SD0714, and SD0755, were identified to be positively infected by BPIV3. The results agreed with what were obtained from RT-PCR on the nasal swab isolation samples. It took the assay merely 92m from sample preparation to data acquisition.

Conclusion A rapid and visualizable method for BPIV3 detection was developed using RT-RPA, magnetic capture, and microfluidic chips. The assay was high on specificity, sensitivity, and applicability for the intended purpose.