Detection of Viable Mycobacterium tuberculosis by Reverse Transcriptase-Strand Displacement Amplification of mRNA

The continued dominance of tuberculosis as a cause of morbidity and mortality (1) has fueled the search for more rapid and reliable means of diagnosis. Numerous systems have now been described for the amplification and detection of DNA or rRNA sequences that are specific for the Mycobacterium tuberculosis complex (2-7). While useful in reducing the amount of time required for definitive diagnosis, these techniques have not proved suitable for monitoring therapeutic efficacy, owing to the persistence of amplifiable nucleic acids for long periods beyond the point of smear and culture conversion (8-14). This presumably reflects both the shedding of dead or dormant bacilli from pulmonary lesions as well as the inherent stability of bacterial DNA and rRNA. In contrast with these nucleic acid targets, bacterial mRNA is typically shortlived with a half-life of only a few minutes (15, 16). Consequently, an mRNA based assay is likely to detect only living organisms and thus be a good indicator of bacterial viability and therefore therapeutic response (9, 12, 13).