本研究旨在建立一种可同时检测非洲马瘟病毒(African horse sickness virus,AHSV)和西尼罗病毒(West Nile virus,WNV)的双重荧光定量RT-PCR检测方法.根据AHSV VP7基因序列和WNV Poly Protein基因的高度保守区域设计特异性引物和探针,优化反应条件及体系,绘制标准曲线,对其特异性、敏感性和重复性进行测定,并采用该方法对临床样品进行检测验证.结果显示:该方法能够同时检测AHSV和WNV,且特异性良好,与马动脉炎病毒、马疱疹病毒1型、马传染性贫血病毒、马腺疫链球菌、马流感病毒等马病核酸均无交叉反应;敏感性高,AHSV和WNV最低检测限均为10 copies·μL-1;组内变异系数为0.04％～0.93％,组间变异系数为0.17％～4.83％,重复性良好;使用该方法对30份马全血样品进行检测,结果均为阴性.本试验建立的检测方法对马属动物检疫、非洲马瘟和西尼罗热的监测与防控具有重要意义.
Establishment and Application of Dual TaqMan Fluorescence RT-PCR Detection Method for African Horse Sickness Virus and West Nile Fever Virus
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 opti-mized,standard curves were established,and the method's specificity,sensitivity,and reproduc-ibility were assessed.Clinical sample testing was conducted to validate the method.Results dem-onstrated that the method could effectively detect both AHSV and WNV with excellent specifici-ty.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.
duplex real-time RT-PCRAfrican horse sickness virusWest Nile virus
万方数据
维普
