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The aim of this study was to evaluate the modified rifampin oligonucleotide (RIFO) macroarray method to detect mutations in the hot-spot region of the rpoB gene, associated with rifampin (RIF) resistance in Mycobacterium tuberculosis. The study sample included 123 strains of M. tuberculosis isolated in the Beijing, China, area in 2002-2005, including 73 RIF-resistant and 40 pansusceptible strains. The genotypic assay successfully identified 91.8% of the RIF-resistant strains, whereas no mutations were found in RIF-susceptible strains. The most frequently detected rpoB mutations were in the codons 516, 526, and 531, together accounting for 74% of RIF-resistant strains. Spoligotyping subdivided all strains into 11 unique profiles and 3 profiles shared by 3, 4, and 103 strains, respectively. The 113 strains belonged to the Beijing family genotypes, defined by the specific spoligotype signature (absence of signals 1-34) and deletion of the RD105 region. The rpoB S531L (TCG→TTG) mutation was found in 57.4% of the RIF-resistant strains of the Beijing genotype. A mutation in the rpoB hot-spot region was found in 51 of the 55 (92.7%) multidrug-resistant strains (i.e., resistant to at least RIF and isoniazid), thus demonstrating the added utility of the modified RIFO method to predict multidrug resistance. The RIFO method is relatively simple to perform and allows straightforward interpretation of results; consequently, it can be used in clinical diagnostic laboratories as a fast complement to phenotypic methods.