Identification of viable and non-viable Mycobacterium tuberculosis in mouse organs by directed RT-PCR for antigen 85B mRNA

Mycobacterium tuberculosis (MTB) the causative organism of tuberculosis can remain dormant as a non-culturable organism, reactivate and cause disease in man and animals. There is a need for proof of viability of such organism s in order to understand the process of reactivation. PCR for bacterial DNA cannot distinguish between viable and non-viable bacilli. We have tested a previously described two tube directed reverse transcriptase polymerase ch ain reaction (RT-PCR) for the detection of mRNA of antigen 85B (Ag85B) of M TB that can distinguish between viable and nonviable organisms. Using a set of external and internal primers for Ag85B, a cDNA amplified product (216 bp) was seen among simulated samples containing only viable cfus at a sensi tivity of >10 and <100 cfu/ml. Eucaryotic DNA rich normal mouse lung homoge nate did not interfere among these samples. The method amplified the 216 bp product also among cfu positive tissues of naturally infected mice. Finall y, in a mouse model of dormancy, direct RT-PCR detected a signal among mult iple tissues that were negative for cfus and hence non-culturable. Ag85B is abundantly secreted by MTB and hyper-expressed under stress conditions. Th us the method to identify its mRNA message may be useful to detect viable b ut dormant bacteria. (C) 2000 Academic Press.