Sypabekova Marzhan, Bekmurzayeva Aliya, Wang Ronghui, Li Yanbin, Nogues Claude, Kanayeva Damira
Laboratory of Biosensors and Bioinstruments, National Laboratory Astana, Nazarbayev University, 53 Kabanbay batyr Avenue, Astana, 010000, Kazakhstan; School of Engineering, Nazarbayev University, 53 Kabanbay batyr Avenue, Astana, 010000, Kazakhstan.
Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR, 72701, USA.
Tuberculosis (Edinb). 2017 May;104:70-78. doi: 10.1016/j.tube.2017.03.004. Epub 2017 Mar 18.
Rapid detection of Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), is important for global control of this disease. Aptamers have emerged as a potential rival for antibodies in therapeutics, diagnostics and biosensing due to their inherent characteristics. The aim of the current study was to select and characterize single-stranded DNA aptamers against MPT64 protein, one of the predominant secreted proteins of Mtb pathogen. Aptamers specific to MPT64 protein were selected in vitro using systematic evolution of ligands through exponential enrichment (SELEX) method. The selection was started with a pool of ssDNA library with randomized 40-nucleotide region. A total of 10 cycles were performed and seventeen aptamers with unique sequences were identified by sequencing. Dot Blot analysis was performed to monitor the SELEX process and to conduct the preliminary tests on the affinity and specificity of aptamers. Enzyme linked oligonucleotide assay (ELONA) showed that most of the aptamers were specific to the MPT64 protein with a linear correlation of R = 0.94 for the most selective. Using Surface Plasmon Resonance (SPR), dissociation equilibrium constant K of 8.92 nM was obtained. Bioinformatics analysis of the most specific aptamers revealed the existence of a conserved as well as distinct sequences and possible binding site on MPT64. The specificity was determined by testing non-target ESAT-6 and CFP-10. Negligible cross-reactivity confirmed the high specificity of the selected aptamer. The selected aptamer was further tested on clinical sputum samples using ELONA and had sensitivity and specificity of 91.3% and 90%, respectively. Microscopy, culture positivity and nucleotide amplification methods were used as reference standards. The aptamers studied could be further used for the development of medical diagnostic tools and detection assays for Mtb.
快速检测结核分枝杆菌（Mtb），即结核病（TB）的病原体，对于全球控制该疾病至关重要。适配体因其固有特性，已成为治疗、诊断和生物传感领域中抗体的潜在竞争对手。本研究的目的是筛选并鉴定针对Mtb病原体主要分泌蛋白之一MPT64蛋白的单链DNA适配体。使用指数富集的配体系统进化（SELEX）方法在体外筛选出对MPT64蛋白具有特异性的适配体。筛选从一个具有随机40个核苷酸区域的单链DNA文库开始。共进行了10轮筛选，通过测序鉴定出17个具有独特序列的适配体。进行斑点印迹分析以监测SELEX过程，并对适配体的亲和力和特异性进行初步测试。酶联寡核苷酸分析（ELONA）表明，大多数适配体对MPT64蛋白具有特异性，最具选择性的适配体的线性相关系数R = 0.94。使用表面等离子体共振（SPR）获得解离平衡常数K为8.92 nM。对最具特异性的适配体进行生物信息学分析，揭示了MPT64上存在保守以及独特的序列和可能的结合位点。通过检测非靶标ESAT-6和CFP-10确定特异性。可忽略不计的交叉反应证实了所选适配体的高特异性。使用ELONA对所选适配体在临床痰液样本上进一步测试，其灵敏度和特异性分别为91.3%和90%。显微镜检查、培养阳性和核苷酸扩增方法用作参考标准。所研究的适配体可进一步用于开发针对Mtb的医学诊断工具和检测方法。
