title: Detection of Mycobacterium tuberculosis directly from spiked human sputum by Q-beta replicase-amplified assay.
abstract: We report on a rapid, sensitive, Q-Beta replicase-amplified nucleic acid 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 method and then washed to neutralize the pH of the cell pellet. The washed sputum pellets were heated at 100 degrees C to inactivate the M. tuberculosis organisms. The heat-inactivated samples were mechanically lysed at 5,000 rpm for 6 min in the GENE-TRAK Sample Processing Instrument in the presence of zirconium oxide beads and a buffer containing guanidine thiocyanate. The released nucleic acid was subjected to the GENE-TRAK Q-Beta replicase-amplified, dual-capture assay. The assay sensitivity was 10(3) purified rRNA targets or 1 CFU of M. tuberculosis spiked into M. tuberculosis-negative human sputum. There was a low level of noise because of the limitations of performing a signal amplification assay in an open system. High levels of other mycobacterial rRNA (approximately 10(7) organisms), including rRNAs of Mycobacterium avium and Mycobacterium gordonae, did not interfere with the sensitivity of the assay.
authors.affiliation: GENE-TRAK, Framingham, Massachusetts 01701.
authors.forename: J S
authors.initials: JS
authors.lastname: Shah
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: D
authors.initials: D
authors.lastname: Buxton
authors.forename: B
authors.initials: B
authors.lastname: Stone
authors.forename: R
authors.initials: R
authors.lastname: Nietupski
authors.forename: D M
authors.initials: DM
authors.lastname: Olive
authors.forename: W
authors.initials: W
authors.lastname: King
authors.forename: J D
authors.initials: JD
authors.lastname: Klinger
mesh_terms: D001431:Bacteriological Techniques / Q000706:statistics & numerical data; D001483:Base Sequence; D015169:Colony Count, Microbial; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005069:Evaluation Studies as Topic; D005784:Gene Amplification; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D011777:Q beta Replicase; D012329:RNA, Bacterial / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D011777:Q beta Replicase