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中国防痨杂志 ›› 2018, Vol. 40 ›› Issue (7): 736-743.doi: 10.3969/j.issn.1000-6621.2018.07.013

• 论著 • 上一篇    下一篇

两种组合显色系统用于分枝杆菌快速药物敏感性试验的性能研究

孙战强()   

  1. 430023 武汉轻工大学生物与制药工程学院(孙战强为客座教授);厦门集庆生物医药科技有限公司(孙战强)
  • 收稿日期:2017-12-27 出版日期:2018-07-10 发布日期:2018-09-07
  • 通信作者: 孙战强 E-mail:sunzhanqiang@126.com
  • 基金资助:
    “十三五”国家科技重大专项(2017ZX10201301)

Development and assessment of the XTT/mPMS combination system for the rapid anti-mycobacteria drug susceptibility testing

Zhan-qiang SUN()   

  1. School of Biology and Pharmaceutical Engineering, Wuhan Polytecnic University ( *Visiting Professor), Wuhan 430023, China
  • Received:2017-12-27 Online:2018-07-10 Published:2018-09-07
  • Contact: Zhan-qiang SUN E-mail:sunzhanqiang@126.com

摘要:

目的 建立一种用于分枝杆菌快速药物敏感性试验(简称“药敏试验”)的3,3'-[1-(苯氨酰基)-3,4-四氮唑]-二(4-甲氧基-6-硝基)苯磺酸钠(XTT)/1-甲氧基-5-甲基酚嗪硫酸甲酯(1-methoxy-PMS,mPMS)组合显色系统(简称“XTT/mPMS”),并加以评估。方法 测试XTT分别与mPMS及2-甲基-1,4-萘醌(2-methyl-1,4-NQ, mNQ)两种中间电子受体的最佳组合浓度、检测线性范围、反应时间、稳定性、细胞毒性、药物干扰作用等特性。以固体培养法作为金标准评估XTT/mPMS用于结核分枝杆菌临床分离株最低抑菌浓度(MIC)的药敏试验性能,并与刃天青显色法结果比较。结果XTT/mPMS和XTT/mNQ两种组合系统中,XTT和中间电子受体的最优组合浓度分别是0.2mmol/L和0.04mmol/L。这两种组合系统中,对耻垢分枝杆菌的显色反应较对牛分枝杆菌和结核分枝杆菌更为迅速(XTT/mPMS组合系统分别为60min、420min、420min;XTT/mNQ组合系统分别为60min、420min、420min)。XTT/mPMS和XTT/mNQ对3种分枝杆菌均为低毒性,能延长其到达稳定期的时间(约滞后12~48h)。XTT/mPMS与7H9液体培养基在37℃恒温中共培养,以及长时间储存中均具有较高的稳定性(37℃共培养可维持8d,25℃条件下稳定性不低于15d,4℃条件下稳定性不低于30d)。以固体培养法为金标准,XTT/mPMS用于结核分枝杆菌临床分离株的快速药敏试验(利福平、异烟肼)检测中,其特异度[分别为100.0%(17/17)和100.0%(13/13)]和敏感度[分别为95.7%(22/23)和92.6%(25/27)]与刃天青显色法[特异度分别为100.0%(17/17)和100.0%(13/13);敏感度分别为100.0%(23/23)和92.6%(25/27)]基本一致。结论 本实验室发现XTT/mPMS组合显色系统稳定、低毒、价格低廉,适合于对分枝杆菌进行快速药敏试验。

关键词: 分枝杆菌属, 分枝杆菌, 结核, 抗药性, 细菌, 微生物敏感性试验, 评价研究

Abstract:

Objective To establish and evaluate a novel colorimetric method (sodium 3,3'-{1-((phenylamino) carbonyl)-3,4-tetrazolium}-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT)/1-methoxy-5-methylphenazinium methylsulfate (1-methoxy-PMS) assay, XTT/mPMS assay) for the rapid anti-mycobacteria drug susceptibility testing.Methods The properties of the XTT/mPMS and XTT/2-methyl-1,4-NQ (mNQ) colorimetric assay, including optimal reagent concentrations, linear range, reaction time, stability, cytotoxicity and drug interference, were assessed. The performance of XTT/mPMS for the rapid drug susceptibility testing of clinical isolated Mycobacteria strains was evaluated taking solid culture method as the gold standard, and the minimum inhibitory concentration (MIC) was determined. The results were compared with the resazurin assay.Results The optimal concentrations of XTT and the electron mediators in XTT/mPMS and XTT/mNQ systems were 0.2 mmol/L and 0.04 mmol/L, respectively. The chromogenic reaction time with Mycobacterium smegmatis was shorter than that with Mycobacterium bovis and Mycobacterium tuberculosis in both systems (XTT/mPMS: 60, 420 and 420 min; XTT/mNQ: 60, 420 and 420 min, respectively). Both systems showed low cytotoxicity to the three mycobacteria, and could prolong the time of reaching the state phase for 12 to 48 hours approximately. The XTT/mPMS system was stable when co-cultured with Middlebrook 7H9 liquid medium at 37 ℃ or when long-term stored at 37 ℃ for 8 d, 25 ℃ for 15 d and 4 ℃ for 30 d. Moreover, using the solid culture method as the gold standard, the specificity of the XTT/mPMS colorimetric method for the rapid drug susceptibility testing (rifampicin and isoniazid) of clinical isolated Mycobacteria strains were 100.0% (17/17) and 100.0% (13/13) and the sensitivity were 95.7% (22/23) and 92.6% (25/27), respectively, which were consistent with the resazurin assay (specificity: 100.0% (17/17) and 100.0% (13/13); sensitivity: 100.0% (23/23) and 92.6% (25/27)).Conclusion We found that the XTT/mPMS chromogenic system is stable, low-toxic and inexpensive, and is well suited for the rapid drug susceptibility testing of Mycobacterium.

Key words: Mycobacterium, Mycobacterium tuberculosis, Drug resistance, bacterial, Microbial sensitivity tests, Evaluation studies