Detection of rifampin resistance in Mycobacterium tuberculosis in a singletube with molecular beacons

Current clinical assays for determining antibiotic susceptibility in Mycoba cterium 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 o f mutations associated with rifampin resistance occur in an 81-by core regi on of the bacterial RNA polymerase gene, rpoB. All mutations that occur wit hin 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 ea ch labeled with a differently colored fluorophore. Together, their target s equences encompass the entire core region. The generation of all five fluor escent 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 abs ence of one of the five fluorescent colors. When 148 M. tuberculosis clinic al isolates of known susceptibility to rifampin were tested, mutations asso ciated with rifampin resistance were detected in 63 of the 65 rifampin-resi stant isolates, and no mutations were found in any of the 83 rifampin-susce ptible isolates. When DNA extracted directly from the sputum of 11 patients infected with rifampin-resistant tuberculosis was tested, mutations were d etected in all of the samples. The use of this rapid assay should enable ea rly detection and treatment of drug-resistant tuberculosis in clinical sett ings.