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.