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

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