結(jié)核分枝桿菌(Mycobacterium tuberculosis)，圖片來自美國疾病防控中心公共健康圖片庫,，Janice Haney Carr。
根據(jù)2012年1月25日發(fā)表在Journal of Clinical Microbiology期刊上的一篇研究論文,，一個國際研究小組優(yōu)化一種讓結(jié)核分枝桿菌(Mycobacterium tuberculosis)在顯微鏡下發(fā)出明亮綠色熒光的策略,。這種篩選活動性結(jié)核病(active tuberculosis)的新方法可能很快給人們提供一種更快度和更簡便的檢測方法。熒光噬菌體(fluorophage)是攜帶熒光報(bào)告基因的細(xì)菌病毒,。研究人員利用該噬菌體去感染從病人肺部咳出的含有結(jié)核菌素(tuberculin)唾液中的分枝桿菌,。幾個小時之內(nèi)，被感染的細(xì)菌表達(dá)報(bào)告基因而且發(fā)出的熒光水平足夠高使得人們在顯微鏡下也能觀察到,。
以前有人試圖構(gòu)建類似地篩選結(jié)核病細(xì)菌的方法,，但是遭遇到報(bào)告基因表達(dá)過低的難題。為了解決這種問題,，研究人員首先從克隆能力較強(qiáng)的噬菌體開始研究,，給它加入一個活性比較強(qiáng)的噬菌體啟動子。這種方法產(chǎn)生的熒光信號是以前報(bào)到的100倍,，而且分枝桿菌陽性的臨床樣品無需對細(xì)菌進(jìn)行培養(yǎng)就可在顯微鏡下檢測到,。此外，這種技術(shù)還允許人們進(jìn)行細(xì)菌耐藥性篩查,。在抗生素存在下,，耐藥性細(xì)菌繼續(xù)發(fā)出熒光，而藥物敏感性細(xì)菌因不能表達(dá)報(bào)告基而不能發(fā)出熒光,。(生物谷：towersimper編譯)
doi:10.1128/JCM.06192-11
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ɸ2GFP10: A high-intensity fluorophage enables detection and rapid drug susceptibility testing of Mycobacterium tuberculosis directly from sputum samples
Paras Jain, Travis E. Hartman, Nell Eisenberg, Max R. O'Donnel, Jordan Kriakov, Karnishree Govender, Mantha Makume, David S. Thaler, Graham F. Hatfull, A. Willem Sturm, Michelle H. Larsen, Preshnie Moodley and William R. Jacobs Jr.
The diagnosis of active tuberculosis (TB) and rapid drug susceptibility testing (DST) at point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterial-specific-fluorophage (φ2GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis (Mtb) in clinical sputum samples. Engineered features distinguishing φ2GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. φ2GFP10 produces a 100-fold increase in fluorescence-per-cell compared to existing reporter-phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. φ2GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampicin and kanamycin from sputum samples yielded results after 12h of incubation with φ2GFP10. Fluorophage φ2GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection, and as a rapid DST.
