DEVELOPMENT OF PCR TECHNIQUES FOR MYCOBACTERIAL DETECTION

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This work's principal focus has been to develop a reliable method for detecting Mycobacterium tuberculosis from clinical specimens through the amplification of mycobacterial DNA using the polymerase chain reaction (PCR). DNA is extracted from clinical samples using a liquid nitrogen-based procedure. Primers specific to the insertion sequence IS6110 are used. This insertion sequence is specific to M. tuberculosis complex and is present in 1 to 20 copies in the M. tuberculosis genome. Internal controls have been developed and are used to detect PCR inhibitors in the samples. Amplification products are visualized using gel electrophoresis. To confirm the presence of the specific amplicons, FCR products are also analyzed by Southern blot and hybridization with chemiluminescent probes. We have been able to demonstrate amplification of M. tuberculosis DNA from sputum specimens. Amplification of M. tuberculosis DNA from buffy coat and tissue specimens is being optimized; we have been able to amplify M. tuberculosis DNA from buffy coats spiked with organism and from a small number of tissue specimens.