Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles
Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles
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DOI：
10.1016/j.mimet.2009.06.009
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发表时间：
2009-09-01
影响因子：
2.2
通讯作者：
Ikonomopoulos, John
中科院分区：
文献类型：
--
作者：
Liandris, Emmanouil;Gazouli, Maria;Ikonomopoulos, John
分枝杆菌感染对经济、人类和动物健康有很大影响。在此，我们提出了一种比色法，依赖于使用金纳米粒子的快速和特异性检测分枝杆菌的发展。而不需要DNA扩增。结果可以通过在酸诱导的AuNP-探针聚集之前和之后的溶液的视觉和/或分光光度比较来记录。互补靶标的存在防止聚集，并且溶液保持粉红色，而在相反的情况下，它变成紫色。应用所提出的方法对分离的细菌产生阳性结果与分枝杆菌分离株和阴性对照。测定的最低检测限被定义为在10 μ l样品体积中稀释的18.75 ng分枝杆菌DNA。为了获得对临床样品的方法的性能的指示，我们应用优化的检测方法来检测鸟分枝杆菌亚种。粪便中的副结核杆菌DNA，与实时PCR比较。两种方法对实时PCR阳性和阴性样本的一致性分别为87.5%和100%。该方法可作为一种高度特异性和灵敏度的筛选工具，以非常简单的方式直接从临床样品中检测分枝杆菌，而不需要高成本的专用设备。本文所述的技术可以发展成一种平台，该平台可以适应许多细菌物种的检测，并且可以容易地适用于高通量和快速筛选样品。(C)2009 Elsevier B. V.保留所有权利。
Mycobacterial infections have a high economic, human and animal health impact. Herein, we present the development of a colorimetric method that relies on the use of gold nanoparticles for fast and specific detection of Mycobacterium spp. dispensing with the need for DNA amplification. The result can be recorded by visual and/or spectrophotometric comparison of solutions before and after acid induced AuNP-probe aggregation. The presence of a complementary target prevents aggregation and the solution remains pink, whereas in the opposite event it turns to purple. The application of the proposed method on isolated bacteria produced positive results with the mycobacterial isolates and negative with the controls. The minimum detection limit of the assay was defined at 18.75 ng of mycobacterial DNA diluted in a sample-volume of 10 mu l. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium avium subsp. paratuberculosis DNA in faeces, in comparison with real-time PCR. The concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%. The proposed method could be used as a highly specific and sensitive screening tool for the detection of mycobacteria directly from clinical samples in a very simple manner, without the need of high-cost dedicated equipment. The technology described here, may develop into a platform that could accommodate detection of many bacterial species and could be easily adapted for high throughput and expedite screening of samples. (C) 2009 Elsevier B.V. All rights reserved.