title: Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease.
abstract: Denaturing high-performance liquid chromatography (DHPLC) is a relatively new technique, which utilizes heteroduplex formation between wild-type and mutated DNA strands to identify point mutations. Heteroduplex molecules are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands. In order to investigate the application of this method for point mutation detection in drug-resistant genes of Mycobacterium tuberculosis, katG, rpoB, embB, gyrA, pncA and rpsL genes, which are responsible for isoniazid, rifampicin, ethambutol, fluoroquinolone, pyrazinamide and streptomycin resistance, respectively, were detected by temperature-mediated DHPLC in 10 multidrug-resistant and 10 drug-susceptible clinical isolates. The DHPLC data were compared with those from a conventional MIC test. The results show that DHPLC is cost-effective with high capacity and accuracy, and is potentially useful for genotypic screening for mutations associated with anti-tuberculosis drug resistance.
authors.affiliation: Henan Provincial Research Institute of Tuberculosis, Zhengzhou, China.
authors.forename: Ruiru
authors.initials: R
authors.lastname: Shi
authors.forename: Koji
authors.initials: K
authors.lastname: Otomo
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamada
authors.forename: Taiga
authors.initials: T
authors.lastname: Tatsumi
authors.forename: Isamu
authors.initials: I
authors.lastname: Sugawara
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004269:DNA, Bacterial; D003851:Deoxyribonucleases / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification*; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes; D013696:Temperature
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D009692:Nucleic Acid Heteroduplexes; D003851:Deoxyribonucleases