DEVELOPMENT OF PCR TECHNIQUES FOR MYCOBACTERIAL DETECTION

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The principal focus of this work has been to develop a reliable method for detection of mycobacteria in clinical specimens by PCR amplification of DNA. Initial attempts were made to use both species and genus specific primer sets, with the goal of enabling detection, and subsequent identification, of all clinically significant mycobacteria. The results with the genus primers were somewhat disappointing, however, with amplification of non-mycobacterial genera (including corynebacteria) presenting a problem in terms of detection format and amplification efficiency. The results with M. tuberculosis-specific amplification have been more encouraging, and we have been able to demonstrate consistent amplification of DNA from sputum specimens containing this organism. We are currently working on developing a colorimetric microtiter plate assay for detection of PCR product, and hope that with this procedure we will be able to determine if acid fast organisms detected in direct smears are M. tuberculosis or not. We have also begun using a liquid-nitrogen-based extraction procedure, which has improved the efficacy of the DNA extraction process from mycobacteria.