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.

Key words: Avian pathogenic Escherichia coli, ISS, ompT, dual-fluorescence quantitative PCR, TaqMan

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,R2=0.99,拷贝数102~109 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,R2=0.997,拷贝数102~ 109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102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。

关键词: 禽致病性大肠埃希菌, ISS, ompT, 双重荧光定量PCR, TaqMan