Identification of rifampin-resistant Mycobacterium tuberculosis strains byhybridization, PCR, and ligase detection reaction on oligonucleotide microchips

Three nerv molecular approaches were developed to identify drug-resistant s trains of Mycobacterium tuberculosis using biochips with oligonucleotides i mmobilized in polyacrylamide gel pads. These approaches are significantly f aster than traditional bacteriological methods. All three approaches-hybrid ization, PCR, and ligase detection reaction-were designed to analyze an 81- bp fragment of the gene rpoB encoding the beta -subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides an d enabled us to identify 30 mutant variants of the rpoB gene within 24 h. T hese variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allel e-specific on-chip PCR, it was possible to directly identify mutations in c linical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a mo del mixture containing 1% of resistant and 99% of susceptible bacteria. Thi s level of sensitivity is comparable to that from the determination of M. t uberculosis drug resistance by using standard bacteriological tests.