荧光PCR熔解曲线法检测结核分枝杆菌耐药基因的结果分析

doi:10.3969/j.issn.1000-6621.2018.09.010

摘要/Abstract

摘要：

目的分析和探讨荧光PCR熔解曲线法对结核病的诊断和耐药检测的应用价值。方法收集2015年9月至2016年12月新疆维吾尔自治区胸科医院的976例疑似结核病患者的痰标本,运用探针荧光PCR熔解曲线法来检测结核分枝杆菌及其对利福平、异烟肼的耐药突变情况。以BACTEC MGIT 960液体培养法及液体药敏试验检测结果为标准,评价探针荧光PCR熔解曲线法鉴定结核分枝杆菌及其耐药突变检测的敏感度、特异度、一致率、Kappa值等。结果以MGIT 960液体培养结果为标准,荧光PCR熔解曲线法鉴定结核DNA的敏感度为85.44%(135/158),特异度为94.01%(769/818),Kappa值为0.75,诊断符合率为92.62%(904/976)。以MGIT 960液体药敏试验结果为标准,荧光PCR熔解曲线法对异烟肼耐药突变检测的敏感度为83.33%(20/24),特异度为94.59%(105/111),Kappa值为0.76,诊断符合率为92.59%(125/135);荧光PCR熔解曲线法对利福平耐药突变检测的敏感度为95.83%(23/24),特异度为95.50%(106/111),Kappa值为0.86,诊断符合率为95.56%(129/135)。结论荧光PCR熔解曲线法检测速度快,对利福平和异烟肼耐药突变检测结果具有良好的敏感度和特异度,可用于临床上对结核分枝杆菌利福平和异烟肼耐药情况的快速筛查。

关键词: 结核, 抗多种药物性, 荧光免疫测定, 分子探针技术, DNA突变分析, 利福平, 异烟肼

Abstract:

Objective To evaluate the value of fluorescence PCR melting curve analysis in Mycobacterium tuberculosis (MTB) identification and drug-resistant TB detection.Methods The sputum specimens from 976 suspected TB patients were collected during September 2015 to December 2016 in Chest Hospital of Xinjiang Uyghur Autonomous Region. MTB and its drug resistance to rifampicin and isoniazid were detected by fluorescence PCR melting curve analysis. The results of MGIT 960 liquid culture method and liquid drug resistance test were stipulated as criterion to evaluate the sensitivity, specificity, agreement rate and Kappa value of melting curve analysis in MTB identification and drug-resistant TB detection.Results The sensitivity, specificity, Kappa value and diagnostic coincidence rate of the melting curve analysis for MTB DNA identification were 85.44% (135/158), 94.01% (769/818), 0.75 and 92.62% (904/976), respectively, taking the result of MGIT 960 liquid culture as gold standard. Taking MGIT 960 liquid drug resistance test as gold standard, the sensitivity, specificity, Kappa value and diagnostic coincidence rate of the melting curve analysis for isoniazid-resistant mutation detection were 83.33% (20/24), 94.59% (105/111), 0.76 and 92.59% (125/135), respectively. Meanwhile, the sensitivity, specificity, Kappa value and diagnostic coincidence rate of the melting curve analysis for rifampin-resistant mutation detection were 95.83% (23/24), 95.50% (106/111), 0.86 and 95.56% (129/135), respectively.Conclusion The probe based melting curve analysis can detect rifampicin-resistant and isoniazid-resistant mutation rapidly and accurately, which can be used for the rapid detection of the multi-drug resistant mutations of MTB.

Key words: Tuberculosis, mutidrug-resistant, Fluroimmunoassay, Molecular probe techniques, DNA mutationcel analysis, Rifampin, Isoniazid

刘艳,古丽比克·木拉提,玛力亚木·阿布力提甫,易星,李君莲,师永欢,刘天剑. 荧光PCR熔解曲线法检测结核分枝杆菌耐药基因的结果分析[J]. 中国防痨杂志, 2018, 40(9): 959-963. doi: 10.3969/j.issn.1000-6621.2018.09.010

Yan LIU,Mulati Gulibike·,Abulitifu Maliyamu·,Xing YI,Jun-lian LI,Yong-huan SHI,Tian-jian LIU. Results analysis on detection of drug resistance gene of Mycobacterium tuberculosis by using fluorescence PCR melting curve method[J]. Chinese Journal of Antituberculosis, 2018, 40(9): 959-963. doi: 10.3969/j.issn.1000-6621.2018.09.010

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参考文献 24

[1]	World Health Organization. Global tuberculosis report 2017. Geneva:World Health Organization, 2017.
[2]	Maitre T, Aubry A, Jarlier V , et al. Multidrug and extensively drug-resistant tuberculosis. Med Mal Infect, 2017,47(1):3-10. doi: 10.1016/j.medmal.2016.07.006 URL
[3]	Yang X, Yuan Y, Pang Y , et al. The burden of MDR/XDR tuberculosis in coastal plains population of China. PLoS One, 2015,10(2):e0117361. doi: 10.1371/journal.pone.0117361 URL pmid: 25689373
[4]	孙敬涛, 华刚, 马丙乾 , 等. 抗痨方辅助化疗药物治疗耐多药肺结核50例临床观察. 中国实验方剂学杂志, 2015,21(8):190-193.
[5]	吕广波 . 控制与降低耐药结核病面临的挑战及防治对策. 医学动物防制, 2016,32(6):640-642.
[6]	沈欣, 宋沈超, 雷世光 , 等. 耐药结核病流行现状及防控对策. 职业与健康, 2017,33(4):566-568.
[7]	Hu S, Li G, Li H , et al. Rapid detection of isoniazid resistance in Mycobacterium tuberculosis isolates by use of real-time PCR-based melting curve analysis. J Clin Microbiol, 2014,52(5):1644-1652. doi: 10.1128/JCM.03395-13 URL
[8]	张文宏, 孙峰 . 以结核病精准诊断与治疗为基础提升我国耐多药结核病防治水平. 中国防痨杂志, 2018,40(2):124-127.
[9]	Coll F, Phelan J, Hill-Cawthorne GA , et al. Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis. Nat Genet, 2018,50(2):307-316. doi: 10.1038/s41588-017-0029-0
[10]	胡春梅 . 荧光定量PCR探针熔解曲线法在结核分枝杆菌耐药基因检测中的应用. 中国防痨杂志, 2016,38(1):38-41. doi: 10.3969/j.issn.1000-6621.2016.01.009 URL
[11]	Liang B, Tan Y, Li Z , et al. Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium tuberculosis by Deep Melt Assay. J Clin Microbiol, 2018, 56(2). pii:e01239-17.
[12]	张立, 吴敏, 侯志丽 . 荧光PCR熔解曲线法快速检测耐多药结核菌的临床应用. 山东医药, 2014,54(36):40-41. doi: 10.3969/j.issn.1002-266X.2014.36.014 URL
[13]	李玉静, 马京华, 张彦坤 , 等. 8种结核病实验室检测方法诊断价值的对比分析. 医学动物防制, 2017,33(1):25-28.
[14]	陆震宇, 杨明芳, 陈纬 . 结核病实验室诊断技术评价及其研究进展. 中国卫生检验杂志, 2017,27(2):301-304.
[15]	沈玉祯 . 结核病实验室诊断技术. 齐齐哈尔医学院学报, 2013,34(4):563-565.
[16]	李辉, 谭耀驹, 李洪敏 , 等. 利福平耐药实时荧光定量核酸扩增检测技术的诊断效果对比研究. 中国防痨杂志, 2014,36(6):472-476. doi: 10.3969/j.issn.1000-6621.2014.06.013 URL
[17]	路丽苹, 陆斌, 刘梅 , 等. GeneXpert MTB/RIF技术在结核病早诊断中的应用. 中国防痨杂志, 2017,39(1):71-75.
[18]	牛海军, 王歌, 李明虎 . GeneXpert MTB/RIF检测在肺结核诊断中的价值评估. 中国防痨杂志, 2017,39(8):829-832.
[19]	孙炳奇, 张娟, 孙娇 , 等. 探针熔解曲线技术检测结核杆菌耐药的研究. 中国医药指南, 2014,12(13):76-77.
[20]	张国钦, 钟达, 陈盛玉 , 等. GenoType线性探针技术检测结核分枝杆菌及其药物敏感性的价值. 结核病与肺部健康杂志, 2016,5(4):267-270. doi: 10.3969/j.issn.2095-3755.2016.04.005 URL
[21]	黄玉平, 屈亚虹, 马振亚 , 等. 应用线性探针技术快速诊断耐多药肺结核效果分析. 中国防痨杂志, 2011,33(11):722-724.
[22]	马晓光, 李辉, 石洁 , 等. 荧光PCR探针熔解曲线法检测结核分枝杆菌耐异烟肼突变. 现代预防医学, 2013,40(22):40-43.
[23]	马艳艳, 李辉, 赵东阳 , 等. 荧光PCR探针熔解曲线法检测结核分枝杆菌耐利福平突变研究. 中国国境卫生检疫杂志, 2011,34(6):451-454.
[24]	李国利, 张灵霞, 王倩 , 等. 结核分枝杆菌临床分离株利福平耐药表型及rpoB基因型分析. 临床和实验医学杂志, 2011,10(21):1649-1652.

荧光PCR熔解曲线法	MGIT 960液体培养(份)		合计 (份)	符合率 (%)	Kappa值	敏感度 (%)	特异度 (%)	阳性预测值(%)	阴性预测值(%)
荧光PCR熔解曲线法	阳性	阴性	合计 (份)	符合率 (%)	Kappa值	敏感度 (%)	特异度 (%)	阳性预测值(%)	阴性预测值(%)
阳性	135(a)	49(b)	184	92.62	0.75	85.44	94.01	73.37	97.10
阴性	23(c)	769(d)	792
合计	158	818	976

荧光PCR熔解曲线法	MGIT 960药敏试验(份)		合计 (份)	符合率 (%)	Kappa值	敏感度 (%)	特异度 (%)	阳性预测值(%)	阴性预测值(%)
荧光PCR熔解曲线法	耐药	敏感	合计 (份)	符合率 (%)	Kappa值	敏感度 (%)	特异度 (%)	阳性预测值(%)	阴性预测值(%)
耐药	20(a)	6(b)	26	92.59	0.76	83.33	94.59	76.92	96.33
敏感	4(c)	105(d)	109
合计	24	111	135

荧光PCR熔解曲线法	MGIT 960药敏试验(份)		合计 (份)	符合率 (%)	Kappa值	敏感度 (%)	特异度 (%)	阳性预测值(%)	阴性预测值(%)
荧光PCR熔解曲线法	耐药	敏感	合计 (份)	符合率 (%)	Kappa值	敏感度 (%)	特异度 (%)	阳性预测值(%)	阴性预测值(%)
耐药	23(a)	5(b)	28	95.56	0.86	95.83	95.50	82.14	99.07
敏感	1(c)	106(d)	107
合计	24	111	135