【摘要】 目的 建立一种检测禽致病性大肠埃希菌(APEC)的双重TaqMan qPCR方法。方法 以APEC保守基因ISS(血清抗性基因)及ompT(保护蛋白基因)为靶标设计特异性引物，以Blunt-ISS及Blunt-ompT质粒标准品为模板，建立双重qPCR检测方法。结果 ISS建立的qPCR方法标准曲线为Y=-3.367+38.518,R~2=0.99,拷贝数10~2～10~9范围内与Ct值有很好的线性关系；ompT建立的qPCR方法标准曲线为Y=-3.314+36.817,R~2=0.997,拷贝数10~2～10~9范围内与Ct值有很好的线性关系。特异性试验显示两种方法与其他鸭病无交叉反应；敏感性实验表明，两种方法对Blunt-ISS及Blunt-ompT的最低检测限均为10~2拷贝数；重复性实验表明，相同条件下建立的两种方法重复性良好；60份临床组织样品检测结果与普通PCR符合率为100%。结论 本研究建立的TaqMan qPCR方法特异性强，灵敏性高，重复性好，适用于临床检测。更多还原

【Abstract】 Objective To establish a dual TaqMan qPCR method for detecting avian pathogenic Escherichia coli（APEC）.Methods Specific primers were designed targeting the conserved genes ISS（serum resistance gene） and ompT（protective protein gene） of APEC. Using Blunt-ISS and Blunt-ompT plasmid standards as templates, a dual qPCR detection method was established.Results The standard curve for the qPCR method targeting ISS was Y=-3.367X+38.518, with R²=0.99, showing a strong linear relationship between the Ct value and copy numbers ranging from 10² to 10⁹. The standard curve for the qPCR method targeting ompT was Y=-3.314X+36.817, with R²=0.997, also demonstrating a strong linear relationship between the Ct value and copy numbers ranging from 10² to 10⁹. Specificity tests confirmed no cross-reactivity with other duck pathogens. Sensitivity experiments indicated that the limit of detection（LOD） for both Blunt-ISS and Blunt-ompT was 10² copies. Repeatability tests showed that both method exhibited excellent reproducibility under identical conditions. Clinical testing of 60 tissue samples revealed 100% concordance with conventional PCR.Conclusion The TaqMan qPCR method established in this study is highly specific, sensitive, reproducible, and suitable for clinical detection.更多还原