Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for molecular detection of M. tuberculosis

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• 作者：Pooria Gill ; Reihaneh Ramezani ; Majid V.-P. Amiri ; Amir Ghaemi ; Taiebeh Hashempour ; Naser Eshraghi ; Mostafa Ghalami and Hossein A. Tehrani
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2006
• 出版时间：8 September 2006
• 年：2006
• 卷：347
• 期：4
• 页码：1151-1157
• 全文大小：257 K

文摘

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-ethylbenzthiazolinsulfonate) substrate. Using this method, as little as 1 × 10² 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.