【摘要】 目的建立生物荧光技术检测结核分枝杆菌ATP含量的方法并观察几种抗结核药物(异烟肼,利福平,乙胺丁醇,链霉素,卡那霉素)的作用。方法结核分枝杆菌标准株H37Rv的菌悬液,经高温裂解,提取ATP,用生物荧光技术检测ATP含量。条件稳定后,观察菌数和ATP浓度的关系,以及加入不同浓度的抗结核药物后1周之内ATP的动态变化。结果菌液加入裂解液后100℃煮沸8min,迅速降至室温,加入荧光素酶,3～5min时测荧光值。按上述步骤提取和测定时菌数和ATP浓度成正比关系。含药液体培养情况:随药物浓度增加,ATP量下降,培养至第7天时,药物的敏感性可以初步进行判断。结论所建立的生物荧光法检测结核分枝杆菌ATP含量,具有简便、快速、敏感、可重复性强等特点,有望成为结核菌体外药敏试验的一种有用的方法。更多还原

【Abstract】 Objective To monitor mycobacterial growth by bioluminescence assay of mycobacterial ATP and to analyze the susceptibility of several antituberculosis drugs(isoniazid,rifampicin,ethambutol,streptomycin,kanamycin).Methods ATP was extracted from the bacterial suspension prepared from mycobacterium tuberculosis H37Rv and measured by bioluminescent assay.The relationship between ATP contents and the number of bacteria was evaluated.The ATP contents of H37Rv inoculated into the Middlebrook 7H9 broth medium containing antituberculosis agents were measured at days of 0,3,5 and 7.Results The highest relative light units(RLU)was obtained when ATP was extracted with lysis buffer at 100℃ boiling for 8 minutes and then decreased quickly to room temperature.3~5 minutes after adding biluminescence enzyme was found to be the proper time for RLU test.The ATP contents correlated well with the number of bacteria.The control culture showed the time-dependent increase in the RLU values,while cultures supplemented with antimicrobial agents reduced their ATP contents concomitant with the concentrations of drugs.Conclusion The bioluminescence assay of mycobacterial ATP established in this work is simple,rapid,sensitive and highly reproducible and may be useful for drug susceptibility assessment in Mycobacterium tuberculosis.更多还原