Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease
Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease
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DOI：
10.1016/j.micinf.2005.06.008
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发表时间：
2006-01-01
影响因子：
5.8
通讯作者：
Sugawara, I
中科院分区：
文献类型：
--
作者：
Shi, RR;Otomo, K;Sugawara, I
变性高效液相色谱（DHPLC）是一种相对较新的技术，它利用野生型和突变DNA链之间的异双工形成来鉴定点突变。异双相分子与同双相分子通过离子对反相液相色谱分离，在特殊的柱基质上对DNA链进行部分热变性。为探讨该方法在结核分枝杆菌耐药基因点突变检测中的应用，采用温度介导的DHPLC对10株多重耐药和10株药敏临床分离株分别进行异烟肼、利福平、乙胺丁醇、氟喹诺酮、吡嗪酰胺和链霉素耐药基因katG、rpoB、embB、gyrA、pncA和rpsL基因的检测。将DHPLC数据与常规MIC测试数据进行比较。结果表明，DHPLC具有高容量和准确性的成本效益，可用于抗结核药物耐药相关突变的基因型筛选。(c) 2005 Elsevier SAS。版权所有。
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. (c) 2005 Elsevier SAS. All rights reserved.