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

doi:10.19982/j.issn.1000-6621.20230202

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

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()

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)

摘要/Abstract

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

中图分类号:

R52
R535

欧喜超, 赵冰, 滕冲, 张思琦, 许晔, 邢睿达, 裴少君, 夏辉, 王胜芬, 范伟兴, 赵雁林. 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[J]. 中国防痨杂志, 2023, 45(11): 1052-1057. doi: 10.19982/j.issn.1000-6621.20230202

Ou Xichao, Zhao Bing, Teng Chong, Zhang Siqi, Xu Ye, Xing Ruida, Pei Shaojun, Xia Hui, Wang Shengfen, Fan Weixing, Zhao Yanlin. Study on the rapid diagnosis of bovine tuberculosis by dual-target real-time fluorescent PCR[J]. Chinese Journal of Antituberculosis, 2023, 45(11): 1052-1057. doi: 10.19982/j.issn.1000-6621.20230202

图/表 2

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引物名称	序列(5'~3')
IS6110上游引物	GAAGAATCCGCTGAGCTGAAG
IS6110下游引物	AAGAAAGCCGACGCGGTC
IS6110探针	FAM-GGACAACGCCGAATTGCGAAG-BHQ1
IS1081上游引物	CCATCTACGACCAGCCCGAC
IS1081下游引物	CGGTGTCGAGGTGCTCGGCCA
IS1081探针	FAM-CGGGTACTCGACGCTCTGACCGA-BHQ1

检测方法	液体培养		敏感度(%,95%CI)	特异度(%,95%CI)
检测方法	阳性(份)	阴性(份)	敏感度(%,95%CI)	特异度(%,95%CI)
Xpert			77.4(58.9~90.4)	73.3(44.9~92.2)
阳性	24	4
阴性	7	11
Xpert Ultra			96.8(83.3~99.9)	40.0(16.3~67.7)
阳性	30	9
阴性	1	6
双靶标荧光PCR			90.3(74.3~98.0)	40.0(16.3~67.7)
阳性	28	9
阴性	3	6

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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

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

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