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中国防痨杂志 ›› 2023, Vol. 45 ›› Issue (11): 1052-1057.doi: 10.19982/j.issn.1000-6621.20230202

• 论著 • 上一篇    下一篇

双靶标实时荧光PCR检测技术用于牛结核病快速诊断的研究

欧喜超1, 赵冰1, 滕冲2, 张思琦3, 许晔3, 邢睿达1, 裴少君4, 夏辉1, 王胜芬1, 范伟兴5, 赵雁林1()   

  1. 1中国疾病预防控制中心结核病预防控制中心,北京 102206
    2北京市东城区疾病预防控制中心结核科,北京 100050
    3厦门大学生命科学学院,厦门 361102
    4北京大学公共卫生学院,北京 100191
    5中国动物卫生与流行病学中心国家动物结核病参考实验室,青岛 266033
  • 收稿日期:2023-06-15 出版日期:2023-11-10 发布日期:2023-11-03
  • 通信作者: 赵雁林,Email:zhaoyl@chinacdc.cn
  • 基金资助:
    国家重点研发计划(2022YFC2305204);西藏自治区重点研发计划(XZ202201ZY0007N-01)

Study on the rapid diagnosis of bovine tuberculosis by dual-target real-time fluorescent PCR

Ou Xichao1, Zhao Bing1, Teng Chong2, Zhang Siqi3, Xu Ye3, Xing Ruida1, Pei Shaojun4, Xia Hui1, Wang Shengfen1, Fan Weixing5, Zhao Yanlin1()   

  1. 1National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
    2Department of Tuberculosis, Beijing Dongcheng District Center for Disease Control, Beijing 100050, China
    3School of Life Sciences, Xiamen University, Xiamen 361102, China
    4School of Public Health, Peking University, Beijing 100191, China
    5Chinese Center for Animal Health and Epidemiology, National Animal Tuberculosis Reference Laboratory, Qingdao 266033, China
  • Received:2023-06-15 Online:2023-11-10 Published:2023-11-03
  • Contact: Zhao Yanlin, Email: zhaoyl@chinacdc.cn
  • Supported by:
    National Key Research and Development Program(2022YFC2305204);Key Research and Development Plan of Tibet Autonomous Region(XZ202201ZY0007N-01)

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摘要：

目的: 针对结核分枝杆菌复合群(Mycobacterium tuberculosis complex,MTBC)IS6110和IS1081基因,开发双靶标实时荧光PCR检测技术并评价其在剖检奶牛病料中进行MTBC快速检测的可行性,为分子检测技术在牛结核病早期诊断领域的推广应用提供基础数据支持。方法: 以IS6110和IS1081为特异靶基因建立MTBC实时荧光PCR检测体系;通过制备并检测含有不同浓度牛分枝杆菌菌悬液的牛肺组织,确定双靶标实时荧光PCR检测体系的检出限;使用21株常见非结核分枝杆菌标准株的DNA作为扩增模板,确定双靶标实时荧光PCR检测MTBC的特异度;对剖检奶牛组织病料进行MGIT 960液体培养、Xpert MTB/RIF(简称“Xpert”)、Xpert MTB/RIF Ultra(简称“Xpert Ultra”)和双靶标实时荧光PCR检测,以液体培养结果为参照标准,分析双靶标实时荧光PCR检测在剖检奶牛病料中检测MTBC的效能。结果: 双靶标实时荧光PCR检测21株非结核分枝杆菌菌株的DNA,均未检测到扩增曲线,MTBC检测特异度为100.0%(21/21)。组织病料液体培养、Xpert、Xpert Ultra和双靶标荧光PCR检测阳性率分别为64.0%(32/50)、56.0%(28/50)、78.0%(39/50)和76.0%(38/50)。双靶标荧光PCR检测阳性率明显高于Xpert检测阳性率,差异有统计学意义(χ2=4.456,P=0.035),双靶标荧光PCR检测阳性率与Xpert Ultra检测阳性率差异无统计学意义(χ2=0.056,P=0.812)。以液体培养结果为参照标准,双靶标荧光PCR检测MTBC的敏感度和特异度分别为90.3%(28/31)和40.0%(6/15);Xpert Ultra检测MTBC的敏感度和特异度分别为96.8%(30/31)和40.0%(6/15)。结论: 双靶标荧光PCR检测技术可用于牛结核病的早期快速诊断,有助于控制人畜共患结核病的流行和传播。

关键词: 结核,牛, 动物疾病, 分子探针技术, 诊断

Abstract:

Objective: A dual-target real-time fluorescent PCR technique was developed for the detection of IS6110 and IS1081 genes of Mycobacterium tuberculosis complex (MTBC), to evaluate the feasibility of MTBC rapid detection in cow samples, and to provide basic data support for the application of molecular detection technology in the early diagnosis of bovine tuberculosis. Methods: MTBC real-time fluorescence PCR system was established with IS6110 and IS1081 as specific target genes. The limit of detection for two-target real-time fluorescent PCR system was determined by preparing and detecting bovine lung tissues containing different concentrations of Mycobacterium bovis suspensions. Using DNA of 21 common standard nontuberculous mycobacteria (NTM) strains as amplification template, the specificity of dual-target real-time fluorescent PCR detection of MTBC was determined. MGIT 960 liquid culture, Xpert MTB/RIF (Xpert), Xpert MTB/RIF Ultra (Xpert Ultra) and dual-target real-time fluorescent PCR were used to detect the tissue samples. Using liquid culture results as a reference standard, the efficacy of dual target real-time fluorescence PCR detection in detecting MTBC in dissected cow diseased materials was analyzed. Results: According to the results of dual-target real-time fluorescent PCR detecting the DNA of 21 NTM strains, no amplification curve was detected, the detection specificity of MTBC was 100.0% (21/21). The positive rates of liquid culture, Xpert, Xpert Ultra, and dual-target real-time fluorescent PCR detection were 64.0% (32/50), 56.0% (28/50), 78.0% (39/50), and 76.0% (38/50), respectively. The positive rate of dual-target real-time fluorescent PCR detection was significantly higher than that of Xpert detection (χ2=4.456, P=0.035). There was no statistically significant difference between the positive rates of dual-target real-time fluorescent PCR detection and Xpert Ultra detection (χ2=0.056, P=0.812). Based on the results of liquid culture experiments, the sensitivity and specificity of dual-target real-time fluorescent PCR for detecting MTBC were 90.3% (28/31) and 40.0% (6/15), and those of Xpert Ultra were 96.8% (30/31) and 40.0% (6/15), respectively. Conclusion: The dual-target real-time fluorescent PCR technique can be used for early diagnosis of bovine tuberculosis, which is helpful to control the epidemic and spread of zoonotic tuberculosis.

Key words: Tuberculosis, bovine, Animal diseases, Molecular probe techniques, Diagnosis

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