Objective A rapid and visual molecular detection method for bovine parainfluenza virus type 3(BPIV3) has been established utilizing technologies that integrate 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, with bovine viral diarrhea virus (BVDV), bovine adenovirus type 3 (BADV3), and bovine infectious rhinotracheitis virus (IBRV) serving as control groups to assess the specificity of this method. The sensitivity of the method was determined by diluting the RNA extraction solution of the BPIV3 standard viral strain by a factor of ten. A total of 56 serum and nasal swab samples from cattle suspected of acute BPIV3 infection were collected. The serum samples were analyzed using this method, while the separated nasal swab samples underwent testing via reverse transcription-PCR (RT-PCR). The methodology and results were evaluated to determine the consistency and practical application of the method.

Results The amplicon size from the RNA extract of the BPIV3 standard strain was 404 bp. No amplicons were detected in the control groups. The sequencing results of the amplicons showed 100% homology with the highly conserved gp3 gene (NC_002161.1) of BPIV3. Molecular detection of RNA extracts from the BPIV3 standard strain at different concentrations revealed the lowest detection limit of this method to be 2.26×10³ copies µL⁻¹.In a study involving 56 serum samples from cattle suspected of being infected with BPIV3 during the acute stage, cattle numbered SD0078, SD0114, SD0319, SD0601, SD0714, and SD0755 were identified as being infected with BPIV3. These findings were consistent with the results obtained from RT-PCR and nasal swab isolation samples detected using this method. Furthermore, the total time required for this method, from sample processing to the acquisition of test results, was 92 min.

Conclusion A rapid and visual molecular detection method for BPIV3 has been established by combining RT-RPA, magnetic capture, and microfluidic chip technology. This method demonstrates good specificity, sensitivity, and applicability.