Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for molecular detection of M-tuberculosis
Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for molecular detection of M-tuberculosis
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DOI：
10.1016/j.bbrc.2006.07.039
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发表时间：
2006-09-08
影响因子：
3.1
通讯作者：
Tehrani, Hossein A.
中科院分区：
文献类型：
--
作者：
Gill, Pooria;Ramezani, Reihaneh;Tehrani, Hossein A.
建立了结核分枝杆菌核酸序列扩增的酶联免疫吸附试验（NASBA-ELISA）。以16 SrRNA为靶点，采用地高辛（Digoxigenin，DIG）标记NASBA的方法扩增细菌RNA，使DIG标记的RNA扩增子积累。将扩增子与非共价固定在链霉亲和素包被的微量滴定板上的特异性生物素化DNA探针杂交。通过添加抗DIG抗体HRP缀合物和2，2-连氮基-二-（3-乙基-苯并噻唑磺酸盐）底物，比色检测RNA-DNA杂交体。使用这种方法，低至1 × 10（2）CFU ml（-1）的M。结核病在不到5小时内被发现。临床标本检测结果的敏感性和特异性分别为85.7%和96%。在扩增和检测M.在存在非靶细菌的情况下检测结核病，证实了该方法的特异性。(c)2006年爱思唯尔公司All rights reserved.
An enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification (NASBA-ELISA) was developed for molecular detection of Mycobacterium tuberculosis, The primers targeting 16S rRNA were used for the amplification of bacterial RNA by the isothermal digoxigenin (DIG)-labeling NASBA process, resulting in the accumulation of DIG-labeled RNA amplicons. The amplicons were hybridized with a specific biotinylated DNA probe which was non-covalently immobilized on streptavidin-coated microtiter plate. The RNA-DNA hybrids were colorimetrically detected by the addition of an anti-DIG antibody HRP conjugate and 2,2-azino-di-(3-ethyl-benzthiazolinsulfonate) substrate. Using this method, as little as 1 x 10(2) CFU ml(-1) of M. tuberculosis was detected within less than 5 h. Results obtained from the clinical specimens showed 85.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method. (c) 2006 Elsevier Inc. All rights reserved.