Rapid detection of Mycobacterium tuberculosis in respiratory samples by transcription-reverse transcription concerted reaction with an automated system

Abstract

The aim of this study was to evaluate the performance of the transcription-reverse transcription concerted (TRC) method for the detection of Mycobacterium tuberculosis complex (MTC) 16S rRNA in clinical respiratory samples for the diagnosis of pulmonary tuberculosis. TRC is a novel method that enables the rapid and the completely homogeneous real-time monitoring of isothermal sequence RNA amplification without any postamplification procedure. The detection limit of the TRC method for MTC was one organism per 100 mul of sputum. The specificity of the method was confirmed by the absence of positive signals for sputum containing 10(6) M. avium or M. kansasii organisms per 100 microl. A total of 201 respiratory samples from patients diagnosed with or suspected of having tuberculosis were tested. Of the 72 MTC culture-positive samples, the TRC method was positive for 52 (sensitivity, 72.2%), whereas the Roche COBAS AMPLICOR PCR was positive for 58 (sensitivity, 80.6%). Both the TRC method and the COBAS AMPLICOR PCR showed no positive identification for any of the 129 culture-negative samples. The percent agreement between the two methods was 95% (191 of 201 samples). The high sensitivity and specificity together with shorter detection time (within 1 h) of the TRC method allow it to be proposed as a useful method for the rapid detection of MTC in respiratory samples.

FIG. 1.

Schematic description of the elementary steps of the TRC method. The progress of the reaction is monitored by measuring the fluorescence intensity of the reaction mixture. dsRNA, double-stranded RNA.

FIG. 2.

Primers and probes used in the TRC method designed for amplification and detection of MTC 16S rRNA (A) and internal control (B). Ex, excitation wavelength; Em, emission wavelength.

FIG. 3.

(A) Fluorescence monitoring of the TRC reaction of the MTC 16S rRNA calibrator. The initial copy numbers of the calibrator are indicated as follows: diamonds, 10⁵; circles, 10⁴; triangles, 10³; squares, 10²; crosses, 0. (B) Initial copy numbers of the calibrator on a logarithmic scale plotted against the time needed to reach the cutoff value of 1.2. Circles indicate the detection time at 520 nm, and diamonds indicate that at 610 nm. The average of the values obtained with triplicates of each sample is plotted.