XTT/mPMS组合显色系统用于抗分枝杆菌药物筛选的性能评估

Evalutation of the performance of the XTT/mPMS colorimetric method for the anti-mycobacteria drug-screening

目的评估一种用于抗分枝杆菌药物高通量筛选的XTT/mPMS组合显色系统。方法评估XTT/mPMS系统的检测限、稳定性、细胞毒性、培养基组分干扰作用以及药物筛选试验的信噪比、Z'因子等性能指标,进行常用抗分枝杆菌药物最低抑菌浓度(MIC)测定,与刃天青显色法(resazurin microtitre assay,REMA)结果比较。结果 XTT/mPMS对分枝杆菌的检测下限为2.4×107CFU/mL,检测上限不同菌种在1.8×108～7.5×108CFU/mL之间。XTT/mPMS和7H9培养基37℃恒温共培养时至少在7 d内保持相对稳定。XTT/mPMS能与7H9-S培养基组分的营养添加剂(OADC)发生非细胞还原反应;其工作浓度(XTT 0.2 mmol/L,mPMS 0.04 mmol/L)对分枝杆菌具有低细胞毒性;XTT/mPMS检测的Z'因子均大于0.8,信噪比较低;分枝杆菌7H9-S液体培养MIC检测5～7 d(快生菌36～48 h)即可获得结果;MIC结果除M.tuberculosis的乙胺丁醇(EMB)有差异外基本与REMA法一致。结论 XTT/mPMS组合显色系统反应快速、稳定、低毒,适合于抗分枝杆菌药物高通量快速筛选。

Objective To evaluate a high throughput XTT/1-methoxy-PMS （XTT/mPMS） colorimetric method for the anti-mycobacteria drug-screening. Methods The performance indexes of the XTT/mPMS colorimetric method, such as the limit of quantitation, stability, cytotoxicity, interference of growth medium components, signal to noise （S/N） ratio and Z＇ factor, were evaluated, and the minimal inhibitory concentrations （ MIC ） of commonly used anti-mycobacteria drugs were determined. The obtained results were compared with those of the resazurin microtitre assay （REMA）. Results The low limit of the XTT/mPMS for the detection of mycobacteria was 2.4 × 10^7 CFU/mL, but the top limit varied with the species of mycobacteria, from 1.8 × 10^8 to 7.5× 10^8 CFU/mL. The XTT/mPMS system was stable at least 7 days when co-cultured with Middlebrook 7H9 liquid medium at 37 ℃. However, XTT/mPMS could produce non-cellular reduction reaction with the nutrition additives such as oleic aeid-albumin-dextrose-catalase （ OADC ） complex in 7H9-S medium. There was low cytotoxicity on myeohaeteria for the working concentration of XTT/mPMS, that is, 0.2 mmol/L of XTT and 0.04 mmol/L of mPMS. The XTT/mPMS system had low S/N ratios and high Z＇ factors （ all above 0.8 ）. In addition, the results of minimal inhibitory concentration（MIC） could be obtained after 5 to 7 days for M. bovis BCG and M. tuberculosis, and after 36 to 48 hours for M. smegmatis. Moreover, the MIC results were similar to those from the REMA except ethambutol （EMB） in the detection of M. tuberculosis. Conclusion The rapid and stable XTT/mPMS colorimetric method with low cytotoxicity and high throughput could be used to screen the anti-mycobacteria drugs.