SINGLE-TUBE, NESTED, REVERSE-TRANSCRIPTASE PCR FOR DETECTION OF VIABLE MYCOBACTERIUM-TUBERCULOSIS

Several problems remain before molecular biology-based techniques, suc h as PCR, are widely accepted for the detection of infectious agents. Among the most formidable of these problems are the inability of the t ests to distinguish between viable and nonviable organisms. We approac hed this problem by using the fact that bacterial mRNA has an extremel y short half-life, averaging only a few minutes. We reasoned that by t argeting bacterial mRNA by a reverse transcriptase PCR (RT-PCR), a pos itive signal would indicate the presence of a recently viable organism . To test our hypothesis, we chose to target the mRNA coding for the u biquitous 85B antigen of mycobacteria. After partially sequencing the gene coding for 85B, we developed primers that were specific for Mycob acterium tuberculosis. In a single-tube, nested, RT-PCR (STN RT-PCR), these primers detected fewer than 40 CFU in spiked sputum samples and as few as 12 CFU in clinical sputum specimens. The sensitivity of STN RT-PCR with smear-negative samples was as good as that of culture, The specificity was 100%, More importantly, when M. tuberculosis was cult ured with and without 1 mu g of isoniazid per ml, this assay could dis tinguish between those cultures which contained the antibiotic and tho se which did not, Subcultures on Lowenstein-Jensen agar confirmed the viability assessments of the STN RT-PCR. Control experiments demonstra ted that isoniazid did not inhibit the RT-PCR. In addition, when an IS 6110-targeted, DNA PCR was used to examine the same samples, all sampl es through 13 days (the last sample) continued to be positive, irrespe ctive of whether isoniazid was present, thereby demonstrating the supe riority of an mRNA target in the detection of mycobacterial viability.