Abstract:

This study aimed to develop a dual-fluorescence quantitative RT-PCR detection method capable of simultaneously identifying African horse sickness virus (AHSV) and West Nile virus (WNV). Specific primers and probes were designed based on highly conserved regions of the AHSV VP7 gene and WNV Poly Protein gene. The reaction conditions and system were optimized, standard curves were established, and the method's specificity, sensitivity, and reproducibility were assessed. Clinical sample testing was conducted to validate the method. Results demonstrated that the method could effectively detect both AHSV and WNV with excellent specificity. No cross-reactivity was observed with equine arteritis virus (EAV), equid Herpesvirus-1 (EHV-1), equine infectious anemia virus (EIAV), Streptococcus equi subsp. zooepidemicus (SEZ), equine influenza virus (EIV) and other equine nucleic acids. The method exhibited high sensitivity, with the lowest detection limits for AHSV and WNV both at 10 copies·μL^-1. Intra-assay coefficients of variation ranged from 0.04% to 0.93%, inter-assay coefficients of variation ranged from 0.17% to 4.83%, demonstrating good reproducibility. Testing 30 equine whole blood samples using this method yielded negative results. The detection method established in this study holds significant importance for equine animal quarantine, monitoring, and control of African horse sickness and West Nile fever.

Key words: duplex real-time RT-PCR, African horse sickness virus, West Nile virus