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  • Title: Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for molecular detection of M. tuberculosis.
    Author: Gill P, Ramezani R, Amiri MV, Ghaemi A, Hashempour T, Eshraghi N, Ghalami M, Tehrani HA.
    Journal: Biochem Biophys Res Commun; 2006 Sep 08; 347(4):1151-7. PubMed ID: 16870140.
    Abstract:
    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 x 10(2) CFU ml(-1) of M. tuberculosis was detected within less than 5h. 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.
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