Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons

Abstract

Current clinical assays for determining antibiotic susceptibility in Mycobacterium tuberculosis require many weeks to complete due to the slow growth of the bacilli. Here we demonstrate an extremely sensitive single-tube PCR assay that takes less than 3 h and reliably identifies rifampin-resistant M. tuberculosis in DNA extracted directly from sputum. Ninety-five percent of mutations associated with rifampin resistance occur in an 81-bp core region of the bacterial RNA polymerase gene, rpoB. All mutations that occur within this region result in rifampin resistance. The assay uses novel nucleic acid hybridization probes called molecular beacons. Five different probes are used in the same reaction, each perfectly complementary to a different target sequence within the rpoB gene of rifampin-susceptible bacilli and each labeled with a differently colored fluorophore. Together, their target sequences encompass the entire core region. The generation of all five fluorescent colors during PCR amplification indicates that rifampin-susceptible M. tuberculosis is present. The presence of any mutation in the core region prevents the binding of one of the molecular beacons, resulting in the absence of one of the five fluorescent colors. When 148 M. tuberculosis clinical isolates of known susceptibility to rifampin were tested, mutations associated with rifampin resistance were detected in 63 of the 65 rifampin-resistant isolates, and no mutations were found in any of the 83 rifampin-susceptible isolates. When DNA extracted directly from the sputum of 11 patients infected with rifampin-resistant tuberculosis was tested, mutations were detected in all of the samples. The use of this rapid assay should enable early detection and treatment of drug-resistant tuberculosis in clinical settings.

Figures

FIG. 1

Comparison of conventional molecular beacons (top diagrams) to wavelength-shifting molecular beacons (lower diagrams). The fluorescence of both conventional and wavelength-shifting molecular beacons is well quenched when the probes are free in solution (left diagrams), yet both types of probes undergo a conformational reorganization and fluoresce brightly when they bind to their target (right diagrams). When a conventional molecular beacon is bound to a target, its fluorophore absorbs energy from the stimulating light, stores the energy for a few nanoseconds, and then emits that energy as bright fluorescent light of a longer wavelength. However, if the particular fluorophore that is chosen for a conventional molecular beacon does not efficiently absorb energy from the stimulating light, then its fluorescence signal will be weak (for example, when blue laser light is used to stimulate the fluorescence of a red fluorophore). In wavelength-shifting molecular beacons, however, the harvester fluorophore is chosen because it efficiently absorbs energy from the stimulating light (for example, fluorescein efficiently absorbs energy from blue light), and the emitter fluorophore (usually orange or red) is chosen because it is able to efficiently accept energy from the harvester fluorophore, store the energy for a few nanoseconds, and then emit that energy as bright fluorescent light at its own characteristic wavelength.

FIG. 2

Location of the target sequence for each of the five molecular beacons on the complementary strands of the 81-bp M. tuberculosis rpoB core region. Probe B (labeled with tetrachlorofluorescein) and Probe E (labeled with fluorescein) were conventional molecular beacons, while Probe A (labeled with Texas red), Probe C (labeled with tetramethylrhodamine), and Probe D (labeled with rhodamine) were wavelength-shifting molecular beacons.

FIG. 3

Detection of mutations that cause rifampin resistance in M. tuberculosis by the amplification of the rpoB core region in the presence of five differently colored molecular beacons. All of the probes hybridized to the amplicons and generated strong fluorescence signals when rifampin-susceptible strains were tested. However, one or two molecular beacons failed to provide a fluorescence signal when rifampin-resistant strains were tested. In all, 148 different M. tuberculosis clinical isolates were tested. The results obtained from six of these isolates are shown.

FIG. 4

Determination of the specificity of the assay. Twenty-six mycobacterial species were tested (representative results from eight of the species are shown). Only M. tuberculosis (strain H37Rv) and the closely related M. africanum (which is a member of the M. tuberculosis group) elicited fluorescence from the four differently colored rpoB probes that were present (a fifth rpoB probe, labeled with fluorescein, was omitted). The reactions also contained primers for the amplification of a region of the 16S rRNA gene that is conserved in all mycobacteria and a fluorescein-labeled molecular beacon that was complementary to that region. The presence of fluorescence from the fluorescein-labeled probe (plotted in green) served as a control signal that confirmed that the absence of the other four colors was due to significant differences between the rpoB sequence of M. tuberculosis and the rpoB sequence in each of the other species and not due to PCR failure.

FIG. 5

Determination of the sensitivity of the assay. Eight PCR assays were initiated with different quantities of DNA obtained from the M. tuberculosis laboratory strain, H37Rv. The amount of DNA added as a template to each of the eight reactions was calculated to be equivalent to the amount of genomic DNA contained in 0, 2, 20, 200, 2,000, 20,000, 200,000, and 2,000,000 bacilli, respectively. Primers were present to amplify the rpoB core region, and probe E was used to detect the amplicons in real time. The results (shown in the left panel) demonstrate that the number of amplification cycles required to generate a detectable fluorescence signal decreases as the number of target molecules initially present in a reaction increases. A control reaction, which did not contain any template DNA, did not give a signal. The results (shown in the right panel) demonstrate that: (i) the threshold cycle is inversely proportional to the logarithm of the number of target molecules initially present, (ii) quantitative results can be obtained over a wide range of target concentrations, and (iii) the assay is sufficiently sensitive to detect as few as two bacilli.

FIG. 6

Determination of the rifampin susceptibility of M. tuberculosis found in sputum samples. Eleven PCR assays were carried out with DNA extracted from sputum obtained from smear-positive patients infected with rifampin-resistant M. tuberculosis. A control reaction contained DNA from the rifampin-susceptible M. tuberculosis laboratory strain, H37Rv. Although all five differently colored molecular beacons gave a fluorescence signal with the rifampin-susceptible control, one of the five fluorescent colors failed to develop in each of the 11 rifampin-resistant samples.