DIRECT-DETECTION OF MYCOBACTERIUM-TUBERCULOSIS IN RESPIRATORY SPECIMENS IN A CLINICAL LABORATORY BY POLYMERASE CHAIN-REACTION

The emergence of epidemic multiple-drug-resistant (MDR) strains of Myc obacterium tuberculosis in conjunction with an increase in the number of reported cases of tuberculosis (TB) represents a major public healt h problem. In light of a recent outbreak of MDR M. tuberculosis at our center, we began the development of a polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary TB using two sets of prime rs, one based on the IS6110 repeated sequence of M. tuberculosis and t he other based on the protein antigen b (PAB). Reaction conditions wer e first optimized as to the appropriate extraction protocol and the co ncentrations of primer pairs, nucleotides, and MgCl2. Following a prel iminary evaluation of the assay with clinical specimens, extraction an d amplification procedures were further modified. PAB and IS6110 prime rs detected between 2 and 23 and 0.023 and 0.23 CFU of M. tuberculosis , respectively, in pooled, M. tuberculosis-negative sputa by our optim ized PCR assay. After routine processing for mycobacteria, 734 specime ns were subsequently amplified. DNA for amplification was obtained by boiling and beating the sediments with Tween 20. For each reaction, DN A (10 mul) was added to an amplification mixture containing 12 pmol of IS6110 primers, 20 pmol of PAB primers, 2 mM MgCl2, 200 muM nucleotid es, and 2.5 U of Taq polymerase and the mixture was then amplified for 40 cycles. The sensitivity and specificity of our PCR assay were 87.2 and 97.7%, respectively. We were unable to interpret the results for seven specimens (1%). In our experience, PCR proved to be a useful rap id diagnostic test for TB in a clinical setting and a valuable epidemi ological tool for determining exposure groups in the hospital setting. Our findings also underscore the need for the systematic optimization of PCR assay conditions.