COMPARISON OF AMPLIFIED Q-BETA REPLICASE AND PCR ASSAYS FOR DETECTIONOF MYCOBACTERIUM-TUBERCULOSIS

Because of the long time required to isolate Mycobacterium tuberculosi s in culture, there is an acute need for simple rapid methods for dire ct detection of M. tuberculosis from human sputum specimens, We have d eveloped and characterized quantitative manual Q beta replicase and PC R assays for M. tuberculosis, The Q beta replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by ampl ification of a replicatable detector probe with Q beta replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plas mid containing an internal deletion in the IS6110 amplicon. Serial dil utions of M. tuberculosis were spiked into sputum and subjected to dig estion and decontamination with N-acetyl-L-cysteine and NaOH, Assay co nditions were optimized for hybridization and sample processing chemis tries in order to maximize sample utilization. Following assay optimiz ation, the sensitivities of the Q beta replicase and PCR assays of spi ked sputum samples were 0.5 and 5.0 CFU per assay reaction, respective ly. The effects of sputum matrix on each assay were examined by testin g 20 patient sputum samples which had been cultured for M. tuberculosi s. The culture-positive samples included smear-positive and smear-nega tive samples. The results of the Q beta replicase assay were not inhib ited by sputum and were in 100% agreement with those of culture, inclu ding detection of 10 culture-positive specimens. However, using an int ernal control plasmid coamplified with each PCR as an indicator, we de tected PCR inhibition in 9 of 20 samples tested, Decreasing the amount of sample assayed in the PCR 24-fold alleviated the inhibitory effect s in all but two specimens, one of which was culture positive. The dec reased sample utilization also resulted in a false-negative result wit h a third specimen which was culture positive for M. tuberculosis. Qua ntitative smear results and Q beta replicase assay estimates of the nu mber of organisms present in these specimens were in close agreement. The Q beta replicase assay performed well in comparison with both cult ure and PCR and should offer a rapid means for detecting and controlli ng infection due to M. tuberculosis.