A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR

Abstract

Species identification of Mycobacterium tuberculosis remains a cumbersome process. We have developed a simple method for treating clinical samples which permits direct polymerase chain reaction (PCR) amplification of mycobacterial target DNA without organic extraction. Samples were boiled for 30 min in TE-Triton, then were subjected to 30 cycles of amplification using primers derived from the repetitive clone pMTb4 developed by Patel and coworkers (1990; Journal of Clinical Microbiology 28, 513). Specificity of amplification was confirmed by hybridization with a specific probe labelled non-isotopically. In a model system consisting of cultured bacilli, this system routinely allows detection of a single organism in a sample. In preliminary studies examining applicability of this method to 96 clinical samples, 74 were positive by both PCR and mycobacterial culture, and eight were negative by both methods. Fourteen samples were negative by culture and positive by PCR, and none were positive by culture and negative by PCR. These results suggest that the PCR method may provide a sensitive alternative to conventional species-specific diagnostic methods, and that non-isotopic labelling can be used to detect hybridization in this assay.