A multiplex polymerase chain reaction assay for genus-, group- and species-specific detection of mycobacteria.
We developed and evaluated a single-step, multiplex polymerase chain reaction (PCR) assay for distinguishing (1) between the Mycobacterium tuberculosis complex (MTBC) and mycobacteria other than tuberculosis (MOTT) and (2) between M. tuberculosis and M. bovis species. The assay targeted the 16S and the 23S rDNA to distinguish between MTBC and MOTT species, and the oxyR gene to distinguish between M. tuberculosis and M. bovis strains. Clinical samples and reference strains (N = 156) comprised 93 strains of M. tuberculosis, 44 of M. bovis, 1 M. africanum strain, and 18 strains representing 9 different species of MOTTs. MOTTs generated only a single PCR product of about 2.5 kilobase; however, all of the MTBC strains produced a 118 base pair (bp) fragment and an additional 270 bp fragment was obtained for M. tuberculosis and M. africanum when the primer pair oxyRTB-2.1/oxyRMT-1 was used. When oxyRTB-2.1/oxyRMB-1 primers were used, the 270 bp fragment was obtained for only M. bovis. The assay needed as little as 1 pg of purified genomic DNA to make a positive identification.
Kurabachew
Mekonnen
M
Centre for International Health, University of Bergen, Norway. mekonnen.kurabachew@cih.uib.no, mekonnenk@hotmail.com
Enger
Øivind
Ø
Sandaa
Ruth-Anne
RA
Skuce
Robin
R
Bjorvatn
Bjarne
B
D000818:Animals; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D004275:DNA, Ribosomal / Q000032:analysis; D016680:Genome, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000145:classification*; D009163:Mycobacterium bovis / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification; D009170:Nontuberculous Mycobacteria / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012494:Sampling Studies; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D004275:DNA, Ribosomal