[1]朱秀兰,邓玉玲,莫丽英,等.锁环探针荧光定量PCR技术检测结核分枝杆菌耐药性及耐药突变基因的应用研究[J].现代检验医学杂志,2026,41(01):111-115+131.[doi:10.3969/j.issn.1671-7414.2026.01.021]
　ZHU Xiulan,DENG Yuling,MO Liying,et al.Application Research of Lock-loop Probe Fluorescence Quantitative PCR Technology for Detecting Drug Resistance and Resistance Mutation Genes in Mycobacterium Tuberculosis[J].Journal of Modern Laboratory Medicine,2026,41(01):111-115+131.[doi:10.3969/j.issn.1671-7414.2026.01.021]
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锁环探针荧光定量PCR技术检测结核分枝杆菌耐药性及耐药突变基因的应用研究()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第41卷
期数:
2026年01期
页码:
111-115+131
栏目:
论著
出版日期:
2026-01-15

文章信息/Info

Title:
Application Research of Lock-loop Probe Fluorescence Quantitative PCR Technology for Detecting Drug Resistance and Resistance Mutation Genes in Mycobacterium Tuberculosis
文章编号:
1671-7414（2026）01-111-06
作者:
朱秀兰，邓玉玲，莫丽英，蒲 荣，陈凤钻，韦学武，卢雪茹，张 帅
广东医科大学附属东莞松山湖中心医院检验科，广东东莞 523000
Author(s):
ZHU Xiulan，DENG Yuling，MO Liying，PU Rong，CHEN Fengzuan，WEI Xuewu，LU Xueru，ZHANG Shuai
Department of Clinical Laboratory, the Affiliated Dongguan Songshan Lake Central Hospital, Guangdong Medical University, Guangdong Dongguan 523000, China
关键词:
结核分枝杆菌; 锁环探针; 荧光定量PCR技术; 利福平; 异烟肼; 耐药性; 耐药基因突变
分类号:
R378.911；Q754；Q503
DOI:
10.3969/j.issn.1671-7414.2026.01.021
文献标志码:
A
摘要:
目的分析锁环探针荧光定量PCR技术检测结核分枝杆菌(MTB)耐药性及耐药突变基因的临床应用价值。方法收集2021年1月～2024年5月广东医科大学附属东莞松山湖中心医院165例涂阳肺结核患者痰液标本，均行痰涂片镜检、MTB培养/鉴定、比例法药敏试验及基于锁环探针的荧光定量PCR技术检测利福平(RFP)、异烟肼(INH)耐药性及耐药基因突变位点。以比例法药敏试验结果为参考标准，评价锁环探针荧光定量PCR技术检测耐药MTB的效能。结果经分离培养证实151株为MTB，比例法药敏试验检出RFP耐药36株，INH耐药42株。锁环探针荧光定量PCR技术检出RFP、INH耐药，与比例法药敏试验检测结果差异无统计学意义（χ2=0.018、0.067，均P>0.05）。以药敏试验为参考，锁环探针荧光定量PCR技术检测RFP耐药的敏感度、特异度、准确度分别为91.67%（33/36）、98.26%（113/115）、96.69%（146/151），检测一致性Kappa值为0.895；检测INH耐药的敏感度、特异度、准确度分别为83.33%（35/42）、95.41%（104/109）、92.05%（139/151），检测一致性Kappa值为0.867。锁环探针荧光定量PCR技术检出RFP耐药菌株rpoB基因突变中96.97%为单一位点突变，以531位点突变为主约占57.58%，其次为526位点突变约占27.27%；检出INH耐药菌株katG/INHA基因突变，以katG315位点突变为主，其中80.56%为katG单一位点突变，16.67%为INHA单一位点突变。结论涂阳肺结核患者应用锁环探针荧光定量PCR技术行痰标本RFP、INH耐药筛查具有较高的诊断效能，可作为临床快速诊断耐药基因位点突变及筛查耐药结核病的补充手段，为临床诊疗提供依据。
Abstract:
Objective To analyze the clinical application value of lock-loop probe fluorescent quantitative polymerase chain reac-tion (PCR) technology for detecting drug resistance and drug resistance mutation genes of Mycobacterium tuberculosis (MTB). Methods Sputum samples from 165 smear-positive pulmonary tuberculosis patients admitted to Dongguan Songshan Lake Central Hospital Affiliated to Guangdong Medical University from January 2021 to May 2024 were collected. Sputum smear microscopy, MTB culture/identification, proportional drug sensitivity test and fluorescent quantitative PCR based on lock-loop probe were used to detect Rifampicin(RFP) and Isoniazid(INH) resistance and gene mutation sites. The efficacy of fluorescent quantitative PCR based on lock-loop probe in detecting drug-resistant MTB was evaluated using proportional drug susceptibility testing results as the reference standard. Results After isolation and culture, 151 strains were confirmed as MTB. RFP resistant 36 strains and INH resistant 42 strains were detected by proportional susceptibility test. The detection of RFP and INH resis-tance by the lock-loop probe fluorescence quantitative PCR showed no significant differences compared with the proportional method (χ2=0.018, 0.067, all P>0.05). Using the drug sensitivity test as a reference, the sensitivity, specificity and accuracy of the lock-loop probe fluorescent quantitative PCR technique for detecting RFP resistance were 91.67% (33/36), 98.26% (113/115) and 96.69% (146/151), respectively, and the Kappa value for detection consistency was 0.895. The sensitivity, specificity and accuracy of INH resistance detection were 83.33% (35/42), 95.41% (104/109) and 92.05% (139/151), respectively, with a con-sistency Kappa value of 0.867. The rpoB gene mutations in RFP resistant strains were detected by lock-loop probe fluorescence quantitative PCR, with 96.97% being single locus mutations. Mutations at 531 locus were predominant (approximately 57.58%) followed by those at 526 locus (approximately 27.27%). Detection of katG/INH A gene mutations in INH resistant strains re-vealed katG mutation at 315 locus as predominant, with 80.56% being katG single site mutations and 16.67% being INH A sin-gle-site mutations. Conclusions The application of lock-loop probe fluorescence quantitative PCR technology for screening RFP and INH resistance in sputum specimens from smear-positive pulmonary tuberculosis patients has a high diagnostic efficiency. It can be used as a supplementary tool for the rapid clinical diagnosis of drug-resistant gene mutations and the screening of drug-re-sistant tuberculosis, providing evidence for clinical diagnosis and treatment.

参考文献/References:

[1] 张慧, 赵雁林, 严俊.我国结核病预防控制进展与挑战[J].中国预防医学杂志, 2025, 26(1):1-7. ZHANG H, ZHAO Y L, YAN J. Progress and challeng-es in tuberculosis prevention and control in China[J]. China Preventive Medicine, 2025, 26(1): 1-7.
[2] GOOSSENS S N, SAMPSON S L, VAN RIE A. Mech-anisms of drug-induced tolerance in Mycobacterium tuberculosis [J]. Clinical Microbiology Reviews, 2020, 34(1): e00141-20.
[3] POULTON N C, ROCK J M. Unraveling the mecha-nisms of intrinsic drug resistance in Mycobacterium tuberculosis [J]. Frontiers in Cellular and Infection Mi-crobiology, 2022, 12: 997283.
[4] 夏珂, 李梦雅, 田晖艳, 等. 多色荧光探针的滚环扩增技术检测结核分枝杆菌耐药基因[J].中华预防医学杂志, 2022, 56(1): 56-62. XIA K, LI M Y, TIAN H Y, et al. Detection of drug resistance genes of Mycobacterium tuberculosis by rolling circle amplification technique with multicolor fluorescent probes [J]. Chinese Journal of Preventive Medicine, 2022, 56(1): 56-62.
[5] 丁磊, 徐俊驰, 邱文娜, 等.结核分枝杆菌耐药机制和治疗的最新研究进展[J].现代检验医学杂志, 2021, 36(2): 1-5, 48. DING L, XU J C, QIU W N, et al. Latest research prog-ress on the drug resistance mechanism and treatment of Mycobacterium tuberculosis[J]. Journal of Modern Laboratory Medicine, 2021, 36(2): 1-5, 48.
[6] 张江峰, 张亚丽, 曾照芳, 等.利用滚环扩增技术快速检测结核分枝杆菌rpoB基因单碱基突变[J].第三军医大学学报, 2013, 35(20): 2155-2158. ZHANG J F, ZHANG Y L, ZENG Z F, et al. Detection of rpoB single base mutations in rifampin-resistant Mycobacterium tuberculosis by rolling circle amplifi-cation[J]. Journal of Third Military Medical University, 2013, 35(20): 2155-2158.
[7] 刁婷婷, 张红吉, 欧阳兵, 等.耐多药结核病实验室检测技术进展[J].检验医学与临床, 2025, 22(1): 131-136. DIAO T T, ZHANG H J, OUYANG B, et al. Advances in laboratory detection techniques for multidrug-resis-tant tuberculosis[J]. Laboratory Medicine and Clinic, 2025, 22(1): 131-136.
[8] 中华医学会, 中华医学会杂志社, 中华医学会全科医学分会, 等.肺结核基层诊疗指南(2018年)[J].中华全科医师杂志, 2019, 18(8): 709-717. Chinse Medical Association, Chinese Medical Journals Publishing House, Chinese Society of General Practice, et al. Guideline for primary care of pulmonary tuber-culosis (2018)[J]. Chinese Journal of General Practi-tioners, 2019, 18(8): 709-717.
[9] CHEN X Y, WANG B, YANG W, et al. Rolling circle amplification for direct detection of rpoB gene mu-tations in Mycobacterium tuberculosis isolates from clinical specimens[J]. Journal of Clinical Microbiology, 2014, 52(5): 1540-1548.
[10] 陈章权, 佃东春, 余路新,等.一种基于锁环探针的荧光定量PCR检测基因突变位点的试剂盒及检测方法:202310019274 [P]. 广东省: CN115927573A, 2023-04-07. CHEN Z Q, DIAN D C, YU L X, et al. A kit and de-tection method for detecting gene mutation sites by fluorescence quantitative PCR based on lock-loop probes :202310019274 [P]. Guangdong Province:CN115927573A, 2023-04-07.
[11] 孙青, 宋华峰, 黄帆.基于基因芯片技术对耐利福平或异烟肼结核分枝杆菌基因突变位点的检测分析[J].抗感染药学, 2023, 20(10): 1108-1112. SUN Q, SONG H F, HUANG F. Detection and analysis of gene mutation sites of Mycobacterium tuberculosis by rifampicin or isoniazid based on gene chip technology[J]. Anti-Infection Pharmacy, 2023, 20(10): 1108-1112.
[12] KANJI A, HASAN R, HASAN Z. E?ux pump as alternate mechanism for drug resistance in Mycobacterium tuberculo-sis [J]. Indian Journal of Tuberculosis, 2019, 66(1): 20-25.
[13] LI H R, YUAN J F, DUAN S J, et al. Resistance and tol-erance of Mycobacterium tuberculosis to antimicrobial agents-How M. tuberculosis can escape antibiotics[J]. WIREs Mechanisms of Disease, 2022, 14(6): e1573.
[14] The Editors. Hetero-resistance: an under-recognised confounder in diagnosis and therapy?[J]. Journal of Medical Microbiology, 2001, 50(12): 1018-1020.
[15] 蒋燕成, 张建明, 陈紫萱, 等.结核分枝杆菌耐药基因突变特征及与耐药水平关系的研究[J].检验医学与临床, 2023, 20(15): 2145-2148. JIANG Y C, ZHANG J M, CHEN Z X, et al. Study on characteristics of drug resistance gene muta-tion in Mycobacterium tuberculosis and its cor-relation with drug resistance level[J]. Laboratory Medicine and Clinic, 2023, 20(15): 2145-2148.
[16] 田丽, 周伟, 黄星, 等.中国异烟肼耐药结核分枝杆菌基因突变特征分析[J].中国防痨杂志, 2022, 44(4):354-361. TIAN L, ZHOU W, HUANG X, et al. Gene mutation analysis of isoniazid-resistant Mycobacterium tubercu-losis in China[J]. Chinese Journal of Antituberculosis, 2022, 44(4): 354-361.
[17] 王悦, 孟湃喆, 于艳红.荧光PCR熔解曲线技术在结核病耐药诊断中的应用价值[J].中国当代医药, 2021, 28(20): 194-197. WANG Y, MENG P Z, YU Y H. Application value of fluorescence PCR melting curve analysis in detecting tuberculosis drug resistance[J]. China Modern Medi-cine, 2021, 28(20): 194-197.
[18] 王丹吉, 刘巧, 卢鹏, 等.基因芯片技术快速检测结核分枝杆菌耐药性的临床应用研究[J].现代预防医学, 2018, 45(11): 2047-2051. WANG D J, LIU Q, LU P, et al. Clinical study on rapid detection of drug resistance of Mycobacterium tuber-culosis by gene chip technology[J]. Modern Preventive Medicine, 2018, 45(11): 2047-2051.
[19] 董启珍, 赵承杰, 吴晓茹.基因芯片技术在新发涂阳肺结核患者结核杆菌菌种鉴定及药敏试验中的应用[J].中华医院感染学杂志, 2021, 31(7): 1024-1028. DONG Q Z, ZHAO C J, WU X R. Application of gene chip technology in strain identification and drug sensitivity test of Mycobacterium tuberculosis in patients with new smear positive pulmonary tuberculosis[J]. Chinese Journal of Nosocomiology, 2021, 31(7): 1024-1028.
[20] SHAO Y, LI Y S, SONG H H, et al. A retrospective co-hort study of isoniazid-resistant tuberculosis treatment outcomes and isoniazid resistance-associated mutations in eastern China from 2013 to 2018 [J]. Journal of Global Antimicrobial Resistance, 2020, 22: 847-853.

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备注/Memo

备注/Memo:
基金项目：东莞市社会发展科技面上项目（编号：20231800937402）。
作者简介：朱秀兰（1973-），女，本科，主任技师，研究方向：微生物检验，E-mail：fjgduvgv@sina.com。
更新日期/Last Update: 2026-01-15