Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare
Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare
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DOI：
10.1128/aac.41.3.570
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发表时间：
1997-03-01
影响因子：
4.9
通讯作者：
Baker, W
中科院分区：
文献类型：
--
作者：
Shawar, RM;Humble, DJ;Baker, W
为了研究荧光素酶快速检测植物提取物中抑菌活性的能力，以重组牛分枝杆菌BCG （rBCG）和表达萤火虫荧光素酶的细胞内分枝杆菌为试验生物，在生物安全等级2的条件下，在96孔微型试管中进行了检测。接种后立即取样对照孔和试验孔，孵育3 d（重组M，胞内）和5 d （rBCG）后，用微孔板光度计测量发光，计算发光相对变化与对照值的百分比，作为替代试验方法，孵育12 d后加入Alamar蓝，目测颜色变化，共检测480个提取物，16个提取物对rBCG有活性；其中7种对重组M细胞内也有活性，活性定义为发光的相对变化小于或等于1% （i,e，大于或等于99%的抑制），并且添加Alamar蓝后持续呈现蓝色，两种方法之间的一致性为99.0%。本研究结果表明，荧光素酶测定快速、准确，有可能大大加快植物提取物体外抑菌活性的评价，该方法可在短时间内筛选大量样品。
The object of this study was to investigate the ability of a rapid luciferase assay to detect antimycobacterial activity in plant extracts, Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacterium intracellulare expressing firefly luciferase were used as the test organisms, Assays were conducted in a 96-well mini-tube format under biosafety level 2 conditions. Control and test wells were sampled immediately after inoculation and after 3 (recombinant M, intracellulare) and 5 (rBCG) days of incubation to measure luminescence with a microplate luminometer, and the relative change in luminescence was calculated as a percentage of control values, As an alternative test method, Alamar blue was added after 12 days of incubation, and changes in color were read visually, a total of 480 extracts were tested, Sixteen extracts were active against rBCG, and of those, seven were also active against recombinant M, intracellulare, With activity defined as a relative change in luminescence of less than or equal to 1% (i,e,, greater than or equal to 99% inhibition) and a persistence of blue color after addition of Alamar blue, there was 99.0% agreement between the two methods, Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimycobacterial activity in plant extracts in vitro, With this method, it is possible to screen a large number of samples in a short period of time.