针对禽致病性大肠埃希菌ISS及ompT基因的TaqMan
双重荧光定量PCR方法的建立及应用

doi:10.3969/ j. issn.1006-6179.2026.01.004

实验动物科学 ›› 2026, Vol. 43 ›› Issue (1): 22-27.DOI: 10.3969/ j. issn.1006-6179.2026.01.004

针对禽致病性大肠埃希菌ISS及ompT基因的TaqMan 双重荧光定量PCR方法的建立及应用

(1.吉林大学动物医学学院,人兽共患病研究所,人兽共患病研究教育部重点实验室,人畜共患传染病重症诊治全国重点实验室, 长春　130062)(2.吉林市丰满区红旗街道综合服务中心,吉林　132013)(3.吉林市丰满区畜牧总站,吉林　132013)

收稿日期:2025-03-17 出版日期:2026-01-28 发布日期:2026-03-05
通讯作者: 赵丽丽(1983—),女,副教授,博士,研究方向为兽医公共卫生。E-mail:zhaolili@jlu.edu.cn。
作者简介:林　蔚(2002—),男,硕士研究生,研究方向为兽医公共卫生。E-mail:2294302409@qq.com。
基金资助:
国家重点研发计划青年科学家项目(2021YFF0703000)。

Development and Application of a TaqMan Dual-Fluorescence Quantitative PCR Method Targeting the ISS and ompT Genes of Avian Pathogenic Escherichia coli (APEC)

(1.Key Laboratory of Zoonosis Research, Ministry of Education, National Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, China) (2.Fengman District Hongqi Sub-District Comprehensive Service Center of Jilin City, Jilin 132013, China) (3.Fengman District Animal Husbandry General Station of Jilin City, Jilin 132013, China)

Received:2025-03-17 Online:2026-01-28 Published:2026-03-05

摘要/Abstract

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

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

中图分类号:

Q95-3

林　蔚, 武景琦, 杨佳美, 郭影成, 李立恒, 赵丽丽. 针对禽致病性大肠埃希菌ISS及ompT基因的TaqMan 双重荧光定量PCR方法的建立及应用[J]. 实验动物科学, 2026, 43(1): 22-27.

Lin Wei, Wu Jingqi, Yang Jiamei, Guo Yingcheng, Li Liheng, Zhao Lili. Development and Application of a TaqMan Dual-Fluorescence Quantitative PCR Method Targeting the ISS and ompT Genes of Avian Pathogenic Escherichia coli (APEC)[J]. Laboratory Animal Science, 2026, 43(1): 22-27.

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针对禽致病性大肠埃希菌ISS及ompT基因的TaqMan 双重荧光定量PCR方法的建立及应用

Development and Application of a TaqMan Dual-Fluorescence Quantitative PCR Method Targeting the ISS and ompT Genes of Avian Pathogenic Escherichia coli (APEC)

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