Detection of Mycobacterium tuberculosis by real-time PCR using pan-mycobacterial primers and a pair of fluorescence resonance energy transfer probes specific for the M. tuberculosis complex.

Abstract

PCR is widely used in clinical laboratories to diagnose pulmonary, extrapulmonary, and disseminated tuberculosis. A multitude of primer pairs have been successfully applied, one of which has been studied most extensively because it is included in the Roche Amplicor MTB assay, the only Food and Drug Administration-cleared PCR-based test for clinical detection of Mycobacterium tuberculosis (1)(2)(3). The test amplifies a 584-bp fragment of the 16s rDNA of all mycobacteria and identifies members of the M. tuberculosis complex (MTC) by hybridization of a specific DNA probe. However, the hybridization step extends the turnaround time of this test, and obligatory license fees render it unaffordable for application in experimental studies or resource-limited settings. Real-time detection technology has made it possible to establish noncommercial, probe-based PCR systems that provide stable operation, low contamination risk, and semiautomated interpretation of results (4)(5)(6)(7)(8)(9)(10)(11). We therefore aimed at adapting the Roche Amplicor assay to a real-time PCR protocol. We did not modify the primers of the Amplicor test because of their demonstrated performance; thus, only the detection probe was adapted to the requirements of real-time PCR. Classic real-time probes [5′-nuclease, “TaqMan” (12)] require cleavage by Taq polymerase (13), which could not be accomplished efficiently in our assay because the amplicon was too long and the only probe binding site specific for MTC was too distant from any of the primers (9)(12)(14). As an alternative approach, we chose a pair of fluorescence resonance energy transfer (FRET) probes that do not have to be cleaved, making them less dependent on the above-mentioned factors (15). On neighboring hybridization to the target DNA, an excited FRET probe system generates long-wavelength fluorescent emission that can be read by a Roche …