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中华临床医师杂志(电子版) ›› 2026, Vol. 20 ›› Issue (04) : 292 -300. doi: 10.3877/cma.j.issn.1674-0785.2026.04.006

基础研究

结核病临床分离株的多技术鉴定及特性分析
魏荣荣1, 葛菲1, 温淑芳1, 赵芯仪1, 郑雨萱1, 车南颖2, 刘毅1,()   
  1. 1 101149 北京,首都医科大学附属北京胸科医院/北京市结核病胸部肿瘤研究所生物样本与数据资源库
    2 101149 北京,首都医科大学附属北京胸科医院/北京市结核病胸部肿瘤研究所病理科
  • 收稿日期:2026-02-13 出版日期:2026-04-30
  • 通信作者: 刘毅
  • 基金资助:
    北京市属医学科研院所公益发展改革试点项目(JYY2023-15)

Multi-technique identification and characterization of clinical isolates of Mycobacterium tuberculosis

Rongrong Wei1, Fei Ge1, Shufang Wen1, Xinyi Zhao1, Yuxuan Zheng1, Nanying Che2, Yi Liu1,()   

  1. 1 Biobank of Beijing Tuberculosis and Thoracic Tumor Research Institute/Beijing Chest Hospital, Capital Medical University, Beijing 101149, China
    2 Department of Pathology, Capital Medical University, Beijing 101149, China
  • Received:2026-02-13 Published:2026-04-30
  • Corresponding author: Yi Liu
引用本文:

魏荣荣, 葛菲, 温淑芳, 赵芯仪, 郑雨萱, 车南颖, 刘毅. 结核病临床分离株的多技术鉴定及特性分析[J/OL]. 中华临床医师杂志(电子版), 2026, 20(04): 292-300.

Rongrong Wei, Fei Ge, Shufang Wen, Xinyi Zhao, Yuxuan Zheng, Nanying Che, Yi Liu. Multi-technique identification and characterization of clinical isolates of Mycobacterium tuberculosis[J/OL]. Chinese Journal of Clinicians(Electronic Edition), 2026, 20(04): 292-300.

目的

建立并验证一套整合传统生化、免疫及质谱技术的结核病临床分离株的标准化鉴定流程,并筛选具有典型特征的菌株。

方法

采用经典的耐热触酶试验、对硝基苯甲酸(PNB)生长试验、硝酸盐还原试验、吐温-80水解试验、尿素酶试验、结核分枝杆菌64KD蛋白(MPT64)抗原检测法和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)等方法对临床样本中获得的分离株进行鉴定分析。采用全基因组测序分析菌株的遗传背景与特征基因,采用微孔板法对菌株进行16种抗结核药物的最小抑菌浓度(MIC)检测。

结果

基础生化实验表明临床分离株符合结核分枝杆菌(M.tb)特性,MPT64抗原胶体金检测结果显示所有菌株为阳性,MALDI-TOF MS鉴定结果显示所有菌株均显示为M.tb特征,置信度达99.9%。全基因组测序分析也显示分离株均为M.tb,分属Lineage 2(北京家族)和Lineage 4(欧美洲谱系),其中16S核糖体核糖核酸(16S rRNA)和热休克蛋白65基因(hsp65)与参考基因组H37Rv的同源性均为100%。MIC结果表明分离株对一线和二线抗结核药物均表现为敏感。传代稳定性观察显示菌落形态、生长特性及关键表型标志均保持稳定。

结论

本研究通过“传统生化表型+免疫学标志+蛋白质组学+基因组学”的多维度验证策略,成功分离出8株背景清晰、特性明确且具有良好体外传代稳定性的M.tb菌株,为实验室质量控制、人员培训及后续标准株评价提供了优质的菌株资源与可靠的方法学基础。

Objective

To establish and validate a standardized protocol integrating traditional biochemical, immunological, and mass spectrometric techniques for the identification of Mycobacterium tuberculosis (M. tb) clinical isolates, and to screen candidate strains with typical characteristics.

Methods

Classical methods, including the heat-stable catalase test, PNB growth test, nitrate reduction test, Tween-80 hydrolysis test, urease test, MPT64 antigen detection, and MALDI-TOF MS, were used to identify clinical isolates obtained from sputum samples. Whole-genome sequencing was employed to analyze the genetic background and signature genes of the strains, and the broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of 16 anti-tuberculosis drugs.

Results

Basic biochemical tests showed that the clinical isolates exhibited characteristics consistent with M. tb. MPT64 antigen colloidal gold detection was positive for all strains, and MALDI-TOF MS identification confirmed that all strains displayed M. tb features with a confidence score of 99.9%. Whole-genome sequencing analysis further revealed that the isolates were M. tb, belonging to Lineage 2 (Beijing family) and Lineage 4 (Euro-American lineage), with 100% homology in the 16S rRNA and hsp65 sequences compared with the reference genome H37Rv. MIC results showed that the isolates were susceptible to all tested first-line and second-line anti-tuberculosis drugs. Subculture stability observation showed that colony morphology, growth characteristics, and key phenotypic markers remained stable over successive passages.

Conclusion

Using a multidimensional verification strategy integrating traditional biochemical phenotyping, immunological markers, proteomics, and genomics, this study successfully isolated and characterized eight M. tb candidate strains with clear genetic backgrounds, well-defined phenotypic characteristics, and good in vitro subculture stability. These strains provide valuable resources for laboratory quality control and personnel training, and and serve as a reliable methodological foundation for future reference strain evaluation.

表1 8株临床分离株的基本信息
图1 候选株在两种培养基上的菌落形态。图a为L-J培养基上的菌落图像;图b为7H10培养基上的菌落图像
表2 8株临床分离株的生化鉴定结果
图2 8株临床分离株及对照菌株的MPT64抗原胶体金法检测结果。图a为阳性对照H37Rv;图b为阴性对照(上:M. fort、下:M.smeg);图c为菌株804(上)与805(下);图d为菌株807(上)与812(下);图e为菌株813(上)与826(下);图f为菌株830(上)与831(下)
图3 8株临床分离株、阳性对照H37Rv及阴性对照M.fortM.smeg MALDI-TOF MS蛋白质指纹图谱。图a为菌株804;图b为菌株805;图c为菌株807;图d为菌株812;图e为菌株813;图f为菌株826;图g为菌株830;图h为菌株831;图i~j为阳性对照H37Rv;图k为阴性对照 M. fort;图l为阴性对照 M.smeg 注:纵坐标为离子强度(相对丰度);横坐标为质荷比(m/z)。经乙醇-甲酸法提取菌体蛋白,采用VITEK® MS PLUS系统采集质谱图(质量范围2000-20 000 Da)。原始图谱经仪器自动去噪、平滑及峰值检测,保留信噪比≥80的特征峰,生成峰值列表(Peak list);基于MATLAB核心运算的MSID高级质谱分析算法,将峰值与种群理念构建的数据库进行加权比对(Binned peak list),经与数据库内各分类模型逐级评分。8株临床分离株(804、805、807、812、813、826、830、831)(图a~h)图谱特征与阳性对照H37Rv(图i~j)高度一致,均鉴定为M.tb(置信度99.9%);阴性对照M.fort(k)、M.smeg(l)图谱特征显著不同,鉴定结果准确,证实了本鉴定体系的分辨力与特异性
表3 8株临床分离株及对照菌株MALDI-TOF MS鉴定结果
表4 8株临床分离株的全基因组测序分析结果
表5 8株临床分离株对16种抗结核药物的MIC及敏感性判定
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