DETECTION OF MYCOBACTERIUM-TUBERCULOSIS BY PCR AMPLIFICATION WITH PAN-MYCOBACTERIUM PRIMERS AND HYBRIDIZATION TO AN MYCOBACTERIUM-TUBERCULOSIS-SPECIFIC PROBE

Nucleic acid amplification techniques such as the PCR are very useful in the rapid diagnosis of infections by Mycobacterium tuberculosis. Ho wever, recent studies have shown that the accuracy of results can vary widely when tests are performed with nonstandardized reagents. We hav e developed a PCR assay for the detection of M. tuberculosis that is b oth rapid and accurate. The assay reagents are standardized and qualit y controlled. False-positive results due to carryover contamination ar e prevented by the incorporation of dUTP coupled with uracil-N-glycosy lase restriction. This assay also employs pan-Mycobacterium amplificat ion primers, allowing for flexibility in the mycobacterial species tha t can be identified from a single amplification reaction. The amplific ation is very sensitive; amplification products generated from as few as three bacteria can be detected by agarose gel electrophoresis, DNAs isolated from 33 of 34 mycobacterial species tested were amplified ef ficiently. Only DNA from Mycobacterium simiae did not amplify. The amp lification is also very specific. Amplification products were generate d only from the DNAs of bacteria in closely related genera such as Cor ynebacterium. The nonmycobacterial amplicons do not pose a problem, as they do not hybridize to mycobacterium-specific probes. Hybridization of amplicons to an M. tuberculosis-specific probe allows for the unam biguous identification of M. tuberculosis complex organisms. The clini cal performance of this PCR assay was evaluated against that of cultur e in 662 respiratory specimens. Sensitivities of 100 and 73.1% were ob tained from smear-positive and -negative respiratory specimens, respec tively. The corresponding specificities were 100 and 99.8%. The high s ensitivity and specificity, coupled with the potential for detecting a wide range of mycobacteria, make this assay a useful tool in the clin ical management of mycobacterial infections.