title: Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for molecular detection of M. tuberculosis.
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.
authors.affiliation: Maebood Clinical and Molecular Laboratory, Tehran, Iran.
authors.forename: Pooria
authors.initials: P
authors.lastname: Gill
authors.forename: Reihaneh
authors.initials: R
authors.lastname: Ramezani
authors.forename: Majid V-P
authors.initials: MV
authors.lastname: Amiri
authors.forename: Amir
authors.initials: A
authors.lastname: Ghaemi
authors.forename: Taiebeh
authors.initials: T
authors.lastname: Hashempour
authors.forename: Naser
authors.initials: N
authors.lastname: Eshraghi
authors.forename: Mostafa
authors.initials: M
authors.lastname: Ghalami
authors.forename: Hossein A
authors.initials: HA
authors.lastname: Tehrani
mesh_terms: D001483:Base Sequence; D004076:Digoxigenin / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D004076:Digoxigenin