To overcome the limitations of traditional detection method for Canine pathogenic Escherichia coli ( CPEC) , a rapid, sensitive, and specific TaqMan probe-based real-time quantitative PCR ( qPCR) method was established for the accurate diagnosis of CPEC. Methods A qPCR detection method targeting the conserved virulence gene eaeA of CPEC was developed. Standard curves were established using serial dilutions to determine the linear range and the limit of detection ( LOD ) .Specificity and repeatability were evaluated. Forty-five clinical samples were used to compare and validate the qPCR method with conventional PCR. Results The established qPCR method exhibited a good linear relationship in the standard curve (R 2 = 0. 99) , with the equation Y = -3. 406x+40. 14. The limit of detection was 10 copies/ μL. No cross-reactivity was observed with other common pathogens.Repeatability experiments showed that both intra-assay and inter-assay coefficients of variation were below 3%. In the detection of clinical samples, the qPCR method identified 15 positive cases, and the consistency between the two methods reached 93. 3%. Conclusion This study successfully established a highly sensitive and specific TaqMan probe-based qPCR detection method, which can provide effective technical support for the early diagnosis and epidemiological monitoring of canine CPEC infections.

Escherichia coli, dog diseases, Real-time polymerase chain reaction, nucleic acid probes, molecular diagnostic techniques

目的 克服犬致病性大肠埃希菌(CPEC)传统检测方法不足,建立快速、灵敏、特异的 TaqMan 探针实时荧光定量 PCR( qPCR)方法,用于 CPEC 的精准诊断。 方法 针对 CEPC 保守毒力基因 eaeA,构建 qPCR 检测方法,梯度稀释建立标准曲线,确定线性范围和最低检出限,评估其特异性、重复性。 用 45 份临床样本与常规 PCR 对比验证。结果 构建的 qPCR 方法标准曲线线性关系良好(R 2 = 0. 99) ,Y = -3. 406x+40.14,最低检出限 10 copies/ μL,对其他常见病原菌无交叉反应。 重复性实验表明,组内与组间变异系数均低于 3%。 临床样本检测中,qPCR 方法检出 15例阳性,两种方法一致性达 93. 3%。 结论 本研究成功建立了一种灵敏度高、特异性强的 TaqMan 探针 qPCR 检测方法,可为犬类感染早期诊断及流行病学监测提供有效技术支持。

大肠埃希菌, 犬疾病, 实时荧光定量 PCR, 核酸探针, 分子诊断技术