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. 1997 Jun;35(6):1477–1483. doi: 10.1128/jcm.35.6.1477-1483.1997

Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay.

J H Smith 1, D Buxton 1, P Cahill 1, M Fiandaca 1, L Goldston 1, L Marselle 1, S Rigby 1, D M Olive 1, A Hendricks 1, T Shimei 1, J D Klinger 1, D J Lane 1, D E Mahan 1
PMCID: PMC229771  PMID: 9163466

Abstract

We have adapted an assay for the direct detection of Mycobacterium tuberculosis using a prototype automated instrument platform in which probes are amplified with Q-beta replicase. The assay was based on amplification of specific detector probe following four cycles of background reduction (reversible target capture) in a closed disposable pack. The assay signal was the time required for fluorescence to exceed background levels (response time [RT]). RT was inversely related to the number of M. tuberculosis rRNA target molecules in the sample. Equivalent signals and noises were observed in assays containing either sputum or buffer. All mock samples containing > or = 10 CFU of M. tuberculosis responded in the assay (average RT, 13.91 min), while most (83%) samples containing as many as 10(7) CFU of Mycobacterium avium gave no response during a 25-min amplification reaction. The samples containing M. avium which did respond had an average RT of 17.04 min. Seventy-five percent (167 of 223) of samples containing no target gave no responses; the remaining 25% had an average RT of 15.53 min. Eighty-three frozen sputum samples were tested to develop a candidate cutoff RT for the assay prior to more extensive clinical testing. After resolution of discrepant results and with a 14-min RT cutoff, 30 of 38 M. tuberculosis-positive samples were positive by the assay; 1 of 45 negative samples responded within 14 min. Assay sensitivity, specificity, and positive and negatives predictive values in this pilot study were 79, 98, 97, and 85%, respectively.

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Selected References

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