Innovation
Focusing on the research and development and application of isothermal nucleic acid amplification technology
Development of a Rapid RT-RAA Diagnostic Method for Bovine Viral Diarrhea Virus Type 1 – Fan Ying
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To address the challenge of rapid on-site diagnosis of bovine viral diarrhea virus (BVDV) and to enhance the speed of emergency response, specific primers and probes were designed based on the conserved 5'-UTR sequence of the BVDV type 1 genome. A recombinase polymerase isothermal nucleic acid amplification method—reverse transcription recombinase aid amplification (RT-RAA)—was established for BVDV type 1, and its specificity, sensitivity, and reproducibility were evaluated. The results showed that this method, performed under isothermal conditions at 39°C, takes only 20 minutes to complete the detection, with a lower limit of detection for BVDV type 1 nucleic acid at 1.2 × 10² copies/μL. The coefficients of variation (CVs) from intra-assay repeatability tests at three different nucleic acid concentration gradients were 3.24%, 9.48%, and 8.79%, respectively—all within the acceptable range of 10.00%, indicating excellent reproducibility. The established method showed no cross-reactivity with swine fever virus, bovine coronavirus, or bovine infectious rhinotracheitis virus. When applied to the analysis of 66 clinical samples, the concordance rate between the RT-RAA method and fluorescence quantitative PCR was 98.48%, with a Kappa value of 0.881 (P < 0.001). These results demonstrate that the RT-RAA method developed in this study exhibits high specificity, high sensitivity, and ease of operation, making it suitable for rapid on-site detection of BVDV type 1.
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