Comparison of an internally controlled, large-volume LightCycler assay for detection of Mycobacterium tuberculosis in clinical samples with the COBAS AMPLICOR assay

Abstract

We present a sensitive and specific assay for reliable and flexible detection of members of the Mycobacterium tuberculosis complex (MTBC) in clinical samples. This real-time PCR assay, which uses the LightCycler 2.0 instrument and 100-mul glass capillaries, can provide a result within 1 h after DNA extraction. The primers amplify a 206-bp fragment of the MTBC 16S rRNA gene. The sensor hybridization probe targets a region highly specific to members of the MTBC. The assay also includes a novel type of internal control that monitors the function of the reaction components and can detect potential inhibitors. Template DNA was extracted by the same procedure used for the COBAS AMPLICOR M. tuberculosis assay, so the LightCycler assay could be directly compared to the COBAS AMPLICOR assay. The LightCycler assay was evaluated with 146 clinical samples of various types. Very good agreement (100% sensitivity, 98.6% specificity) could be shown between the LightCycler and COBAS AMPLICOR assays. Specificity was checked with a panel of nontuberculous mycobacteria, as well as a large panel of bacterial and fungal organisms.

FIG. 1.

Alignment of LightCycler primers and probes with M. tuberculosis, the ICO, and different NTM. BX842576, M. tuberculosis; X52918, M. avium; X15916, M. kansasii; X52930, M. malmoense; X52924, M. scrofulaceum; X52931, M. simiae; AF547970, M. terrae; AY215232, M. celatum; pos., position.

FIG. 2.

Specificity of the LightCycler M. tuberculosis assay. Curves: 1, M. avium; 2, M. kansasii; 3, M. malmoense; 4, M. terrae; 5, M. fortuitum; 6, C. pseudodiphtheriticum; 7, N. brasiliensis; 8, negative sample (sterile water copurified with the samples); 9, M. tuberculosis. Panels: A, fluorescence signals obtained during amplification; B, T_m analysis. The ICO amplification product has a melting peak at about 48°C.

FIG. 3.

Effects of different concentrations of an inhibitor (NALC-NaOH solution) on the amplification of the target and ICO. All reaction mixtures (in 20-μl capillaries) contained 125 copies of ICO. Curves 1 to 5, M. tuberculosis DNA plus 2.5 μl of NALC-NaOH solution. Each capillary contained a different dilution of NALC-NaOH (in sterile water) to produce different concentrations of inhibitor. The dilutions of NALC-NaOH used in the assay were 1:5 (capillary 1), 1:10 (capillary 2), 1:15 (capillary 3), 1:25 (capillary 4), and 1:30 (capillary 5). Capillary 6 was a negative control (sterile water). Panels: A, fluorescence signals obtained during amplification; B, T_m analysis.