Establishment of a Rapid Detection Method for Canine Coronavirus Using Recombinant Enzyme and Polymerase Mediated...

To establish a rapid detection method for canine coronavirus based on recombinant enzyme and polymerase mediated isothermal amplification technology. The 3 'UTR conserved sequence gene fragment of the canine coronavirus genome was used as the target sequence, and a recombinant plasmid was constructed as the standard. Primers and fluorescent probes were designed and synthesized. Primer screening was performed using basic nucleic acid amplification, and a RAPID reaction system was established. Select plasmid standards with different copy numbers as RAPID amplification templates and evaluate their detection sensitivity. Select common canine virus genomes such as CPV, CPIV, CIV, CDV, CAV, ICHV, etc. as amplification templates to evaluate their detection specificity. Select plasmid standards from the same batch and different batches as RAPID amplification templates, and analyze the coefficient of variation within and between groups. Select 40 positive and 40 negative clinical samples of CCV isolated from different clinical stages and identified by RT-PCR as RAPID amplification templates, and analyze their detection consistency rate. The results showed that a RAPID reaction system was established using optimized CCV-F2/CCV-R1 primers, CCV-P probes, and pTCCV3 recombinant plasmid standards. A clear amplification curve was observed within 15 min, and the sensitivity for detecting CCV was 10 copies/μL; No cross reactivity with CPV, CPIV, CIV, CDV, CAV, ICHV, with strong specificity; The coefficient of variation (CV) within and between groups is less than 2.00%, indicating good reproducibility; Compared with RT-PCR, the overall consistency rate of using RAPID method for rapid detection of CCV clinical samples is 96.25%, with higher accuracy. The rapid detection method of canine coronavirus based on recombinant enzyme and polymerase mediated isothermal amplification technology successfully established in this study has superior detection performance and can be used as a CCV rapid detection kit, providing technical reference for on-site rapid detection of canine coronavirus disease and diagnosis of other related canine diseases.