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

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 >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.

Performance of an automated Q-beta replicase amplification assay for Mycobacterium tuberculosis in a clinical trial

“…Reagents. Oligo(dT) 14 -and oligo(dC) 25 -derivatized magnetic particles, capture probes, and RNA detector probes were prepared as described elsewhere (38)(39)(40). All reagents for testing a single sample (RTC, amplification, and detection) were packaged in a closed disposable Surlyn test pack, labeled in bar code form to include information concerning reagent performance characteristics (40).…”

“…After sampling for acid-fast microscopy, this second aliquot was heated at 100°C for 15 min and was then mechanically disrupted in a proprietary sample processing device. These steps took approximately 21 min and are described elsewhere (39,40,51). Previously cultured (negative) sputum aliquots were treated as described above and were then spiked with serial dilutions of M. tuberculosis cells for use in establishing the sensitivity of the assay.…”

“…The prototype instruments were manufactured by Wilj Division, Integrated Technologies, Ltd., Ashford, United Kingdom. The Galileo instrument platform is described in greater detail elsewhere (30,40).…”

“…Amplification data analysis. The Galileo instrument processed each test pack through four rounds of RTC, after which the probe amplification reaction was initiated by the addition of Q-Beta replicase enzyme and nucleoside triphosphates in an appropriate reaction mixture containing propidium iodide (40).…”

“…We have previously described RTC and basic Q-Beta replicase amplification for several infectious disease agents (see the references cited in references 2, 9, 26, 32, 37, 38, 40, 44, and 51). A prototype instrument platform (Galileo) has been developed to totally automate all RTC and amplification and detection steps inside a closed disposable test pack requiring minimal operator intervention or specialized training (30,40). Following sample processing, all assay steps were performed in the disposable test pack, which was designed to limit contamination from replicable RNA, to provide convenient packaging to store all reagents, and to control potential biohazards.…”