全细菌SELEX法筛选粪肠球菌特异性适配体

肠球菌（Enterococcus）是内源性和外源性医院感染的第二大病原菌，检出率仅次于大肠杆菌，从分子水平上发展靶标的高亲和力分子探针对肠球菌的识别和检测具有非常重要的意义。本研究以粪肠球菌为靶标，运用全细菌指数富集的配体系统进化技术（whole-bacteria systematic evolution of ligands by exponential enrichment, whole-bacteria SELEX），从全长为79个核苷酸包含35个随机碱基序列的单链DNA文库中筛选与靶标高亲和力、高特异性结合的适配体，利用荧光分析法监控筛选过程中不同轮次所得次级文库与粪肠球菌的结合力，经12轮筛选和克隆测序，获得了39条适配体序列。进一步对筛选得到的适配体进行序列比对、二级结构分析、流式细胞分析、解离常数（Kd）测定及特异性验证，最终获得一条与粪肠球菌能特异性结合的适配体Apt 21，其Kd值为549.2 ± 147.4 nmol/L。该适配体可作为粪肠球菌检测的识别元件，为建立基于适配体的新型粪肠球菌检测方法奠定了基础。

Screening and Characterization of Enterococcus faecalis-pecific Aptamers through Whole-bacteria SELEX

Enterococcus is the second most common pathogen of endogenous and exogenous nosocomial infections, whose isolation rate is only lower than that of Escherichia coli. It is of great significance to develop molecular probes with high affinity for Enterococcus, which can be used in the identification and detection of Enterococcus. In this study, aptamers against Enterococcus faecalis were screened by whole-bacteria systematic evolution of ligands by exponential enrichment (whole-bacteria SELEX) from a random single-stranded DNA (ssDNA) library with the length of 79 nucleotides containing 35 random bases. During the SELEX process, the affinity of ssDNA sub-library toward E. faecalis was evaluated by fluorescence analysis. After 12 rounds of selection, cloning and sequencing, overall 39 aptamers were screened. Then sequence alignment, secondary structure prediction, flow cytometry analysis, dissociation constant (Kd) determination and specificity evaluation were carried out. Finally, an aptamer named Apt 21 was picked as the optimal aptamer of E. faecalis, with a Kd value of 549.2 ± 147.4 nmol/L. It can be used as the recognition element for E. faecalis to construct novel detection methods for E. faecalis.