Rapid detection and identification of mycobacteria by combined method of polymerase chain reaction and hybridization protection assay

A new method for the rapid detection and identification of mycobacteria, combining polymerase chain reaction (PCR) with DNA probe, was developed. The mycobacterial 16S rRNA encoding gene was amplified by nested PCR, and the PCR product was identified by hybridization protection assay using acridinium ester labelled DNA probe. The optimum temperature for hybridization of PCR product and DNA probe was estimated as 55°C. The specificity of the combined method was excellent in the detection and identification of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare complex (MAC). The detection limit was 10 fg DNA for M. tuberculosis , and 100 fg DNA for MAC. Results of preliminary clinical investigation of this method using 207 clinical specimens demonstrated an efficient detection and identification of M. tuberculosis and MAC. These results indicate that the new combined method may be a useful test for the rapid detection and identification of M. tuberculosis and MAC in clinical specimens.