Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease.
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.
Shi
Ruiru
R
Henan Provincial Research Institute of Tuberculosis, Zhengzhou, China.
Otomo
Koji
K
Yamada
Hiroyuki
H
Tatsumi
Taiga
T
Sugawara
Isamu
I
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
D000995:Antitubercular Agents; D004269:DNA, Bacterial; D009692:Nucleic Acid Heteroduplexes; D003851:Deoxyribonucleases