title: Capillary electrophoresis-single strand conformation polymorphism for the detection of multiple mutations leading to tuberculosis drug resistance.
abstract: Drug resistant tuberculosis (TB) is a major health problem in both developed and developing countries. Mutations in the Mycobacterium (M.) tuberculosis bacterial genome, such as those to the rpoB gene and mabA-inhA promoter region, have been linked to TB drug resistance in against rifampicin and isoniazid, respectively. The rapid, accurate, and inexpensive identification of these and other mutations leading to TB drug resistance is an essential tool for improving human health. Capillary electrophoresis (CE) single strand conformation polymorphism (SSCP) can be a highly sensitive technique for the detection of genetic mutation that has not been previously explored for drug resistance mutations in M. tuberculosis. This work explores the potential of CE-SSCP through the optimization of variables such as polymer separation matrix concentration, capillary wall coating, electric field strength, and temperature on resolution of mutation detection. The successful detection of an rpoB gene mutation and two mabA-inhA promoter region mutations while simultaneously differentiating a TB-causing mycobacteria from a non-TB bacteria was accomplished using the optimum conditions of 4.5% (w/v) PDMA in a PDMA coated capillary at 20°C using a separation voltage of 278 V/cm. This multiplexed analysis that can be completed in a few hours demonstrates the potential of CE-SSCP to be an inexpensive and rapid analysis method.
authors.affiliation: University of Arkansas, Department of Biomedical Engineering, Fayetteville, AR 72701, United States.
authors.forename: Sowmya
authors.initials: S
authors.lastname: Krothapalli
authors.forename: Michael K
authors.initials: MK
authors.lastname: May
authors.forename: Christa N
authors.initials: CN
authors.lastname: Hestekin
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024881:Drug Resistance, Bacterial; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000235:genetics; D018807:Polymorphism, Single-Stranded Conformational; D011401:Promoter Regions, Genetic; D012293:Rifampin / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010088:Oxidoreductases; C085257:InhA protein, Mycobacterium; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D007538:Isoniazid; D012293:Rifampin