DESCRIPTION: (Adapted from the applicant s abstract) The aim of this proposal is to develop high density oligonucleotide arrays which detect and characterize mutations conferring rifampin and rifabutin resistance. Additionally, this array will be used to explore establishing a database consisting of Mycobacterium single nucleotide polymorphisms (SMPs) and hybridization patterns (fingerprints) which will be capable of speciating among Mycobacterium species. In Phase I, they will 1) redesign and synthesize high density arrays which will sequence 700 base pairs of the rpoB gene of Mycobacterium tuberculosis, 2) design specialized tiling arrays on the same chip which will permit unambiguous detection and characterization of insertion and deletion rifampin/rifabutin conferring mutations, 3) resequence the 700 base pair region of rpoB genes from 10 isolates of each of 8 non-tuberculosis, clinically important Mycobacterium species using conventional dideoxynucleotide and high density array-based sequencing. During Phases II and III, previously characterized and uncharacterized rpoB genes from clinical isolates will serve as model targets to test, retrospectively and prospectively, the performance characteristics of the rpoB chip for the detection of drug resistance and speciation. Finally, they will expand the sequence and fingerprint database of this gene (with the analysis of an additional 40 isolates from each of the 8 Mycobacterium species) and include gene sequences for the detection of isoniazid and ethionamide resistance.National Institute of Allergy and Infectious Diseases (NIAID)
