Selection and characterization of DNA aptamers specific for Listeria species
Selection and characterization of DNA aptamers specific for Listeria species
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DOI：
10.1016/j.ab.2014.05.006
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发表时间：
2014-08-15
影响因子：
2.9
通讯作者：
Jaykus, Lee-Ann
中科院分区：
文献类型：
--
作者：
Suh, Soo Hwan;Dwivedi, Hari P.;Jaykus, Lee-Ann
对李斯特菌属具有结合亲和力的单链（ss）DNA适体使用全细胞SELEX（通过指数富集的配体系统进化）方法选择。单核细胞增生李斯特菌细胞在37 ℃下生长，并在对数中期或稳定期早期收获，以用作SELEX中的靶标。共鉴定了10个独特的适体序列，6个与对数期细胞相关，4个与稳定期细胞相关。使用流式细胞术测定适体的结合亲和力，范围为10%至44%。进一步研究了具有高结合亲和力的四种候选物，发现当针对李斯特菌属内的五种不同物种进行筛选时，其显示出属特异性结合亲和力。使用与流式细胞术相结合的连续结合测定，确定三种适体（LM 6 -2、LM 12 -6和LM 12 -13）结合至一个表观细胞表面部分，而第四种适体（LM 6 -116）似乎结合至不同的细胞表面区域。这是第一个研究，其中SELEX针对不同生长阶段的细菌细胞。当一起使用时，结合不同细胞表面部分的适体可以增加未来捕获和检测测定的分析灵敏度。(C)2014爱思唯尔公司All rights reserved.
Single-stranded (ss) DNA aptamers with binding affinity to Listeria spp. were selected using a whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Listeria monocytogenes cells were grown at 37 C and harvested at mid-log phase or early stationary phase to serve as the targets in SELEX. A total of 10 unique aptamer sequences were identified, six associated with log phase cells and four with stationary phase cells. Binding affinity of the aptamers was determined using flow cytometry and ranged from 10% to 44%. Four candidates having high binding affinity were further studied and found to show genus-specific binding affinity when screened against five different species within the Listeria genus. Using sequential binding assays combined, with flow cytometry, it was determined that three of the aptamers (LM6-2, LM12-6, and LM12-13) bound to one apparent cell surface moiety, while a fourth aptamer (LM6-116) appeared to bind to a different cell surface region. This is the first study in which SELEX targeted bacterial cells at different growth phases. When used together, aptamers that bind to different cell surface moieties could increase the analytical sensitivity of future capture and detection assays. (C) 2014 Elsevier Inc. All rights reserved.