Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria.
Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique for nucleic acids (NA), was investigated for the species-specific identification of mycobacteria. A set of primers was selected from a highly conserved region of the 16S rRNA sequence of mycobacteria sandwiching a variable sequence to perform amplification of mycobacterial RNA. Species-specific probes for the M. tuberculosis complex, M. avium-paratuberculosis, M. intracellulare and M. leprae were hybridized in-solution with the amplified nucleic acids of 10 pathogenic mycobacteria and 11 closely related bacteria, as well as with human-derived NA in an enzyme-linked gel assay (ELGA). Each probe was shown to hybridize specifically to the amplified single-stranded RNA of the corresponding species. Thirty-two clinical isolates of M. tuberculosis strains from different parts of the world were correctly identified by NASBA using the M. tuberculosis-complex-specific probe. In combination with the ELGA, NASBA could identify mycobacteria rapidly, i.e. in less than 6 h. The relative simplicity and rapidity of this technique makes it an attractive tool for species-specific identification of mycobacteria.
van der Vliet
G M
GM
Royal Tropical Institute, N.H. Swellengrebel Laboratory of Tropical Hygiene, Amsterdam, The Netherlands.
Schukkink
R A
RA
van Gemen
B
B
Schepers
P
P
Klatser
P R
PR
D001483:Base Sequence; D004275:DNA, Ribosomal / Q000235:genetics; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics* / Q000302:isolation & purification*; D009162:Mycobacterium avium / Q000235:genetics / Q000302:isolation & purification; D009166:Mycobacterium leprae / Q000235:genetics / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity
D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S