Zimenkov D V, Kulagina E V, Antonova O V, Surzhikov S A, Bespiatykh Iu A, Shitikov E A, Il'ina E N, Mikhaĭlovich V M, Zasedatelev A S, Griadunov D A
Mol Biol (Mosk). 2014 Mar-Apr;48(2):251-64.
Steadily growing resistance of the tuberculosis causative agent towards a broad spectrum of anti-tuberculosis drugs calls for rapid and reliable methods for identifying the genetic determinants responsible for this resistance. In this study, we present a biochip-based method for simultaneous identification of mutations within rpoB gene associated with rifampin resistance, mutations in katG, inhA, ahpC genes responsible for isoniazid resistance, mutations within the regions of gyrA and gyrB genes leading to fluoroquinolones resistance, and mutations in the rrs gene and the eis promoter region associated with the resistance to kanamycin, capreomycin and amikacin. The oligonucleotide microchip, as the core element of this assay, provides simultaneous identification of 99 mutations in the format "one sample--one PCR--one microchip", and it makes it possible to complete analysis of multi-drug-resistant and extensively drug-resistant tuberculosis within a single day. The tests on 63 Mycobacterium tuberculosis clinical isolates with different resistance profiles using the developed approach allows us to reveal the spectrum of drug-resistance associated mutations, and to estimate the significance of the inclusion of extra genetic loci in the determination of M. tuberculosis drug resistance.
结核病原菌对多种抗结核药物的耐药性不断增强，这就需要快速可靠的方法来鉴定导致这种耐药性的遗传决定因素。在本研究中，我们提出了一种基于生物芯片的方法，用于同时鉴定与利福平耐药相关的rpoB基因内的突变、与异烟肼耐药相关的katG、inhA、ahpC基因中的突变、导致氟喹诺酮耐药的gyrA和gyrB基因区域内的突变，以及与对卡那霉素、卷曲霉素和阿米卡星耐药相关的rrs基因和eis启动子区域内的突变。作为该检测方法核心元件的寡核苷酸微芯片，以“一个样本 - 一次PCR - 一张微芯片”的形式同时鉴定99种突变，并且能够在一天内完成对耐多药和广泛耐药结核病的分析。使用所开发的方法对63株具有不同耐药谱的结核分枝杆菌临床分离株进行检测，使我们能够揭示与耐药相关的突变谱，并评估在确定结核分枝杆菌耐药性时纳入额外基因位点的重要性。
