DETECTION OF MYCOBACTERIUM-TUBERCULOSIS DIRECTLY FROM SPIKED HUMAN SPUTUM BY Q-BETA REPLICASE-AMPLIFIED ASSAY

We report on a rapid, sensitive, Q-Beta replicase-amplified nucleic ac id hybridization assay for the detection of Mycobacterium tuberculosis directly from spiked human sputum, Specimens were processed by either an N-acetyl-L-cysteine-NaOH or a 2% NaOH digestion-decontamination me thod and then washed to neutralize the pH of the cell pellet. The wash ed sputum pellets were heated at 100 degrees C to inactivate the M, tu berculosis organisms, The heat-inactivated samples were mechanically l ysed at 5,000 rpm for 6 min in the GENE-TRAK Sample Processing Instrum ent in the presence of zirconium oxide beads and a buffer containing g uanidine thiocyanate. The released nucleic acid was subjected to the G ENE-TRAK Q-Beta replicase-amplified, dual-capture assay, The assay sen sitivity was 10(3) purified rRNA targets or 1 CFU of M. tuberculosis s piked into M. tuberculosis-negative human sputum. There was a low leve l of noise because of the limitations of performing a signal amplifica tion assay in an open system, High levels of other mycobacterial rRNA (approximately 10(7) organisms), including rRNAs of Mycobacterium aviu m and Mycobacterium gordonae, did not interfere with the sensitivity o f the assay,