使用SELEX方法筛选和鉴定用于检测ε毒素的DNA纳米结构适体
Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for Detection of Epsilon Toxin.
作者信息
Shafiei Nafiseh, Mahmoodzadeh Hosseini Hamideh, Amani Jafar, Mirhosseini Seyed Ali, Jafary Hanieh
机构信息
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
出版信息
Iran J Pharm Res. 2023 Dec 8;22(1):e140505. doi: 10.5812/ijpr-140505. eCollection 2023 Jan-Dec.
BACKGROUND
Epsilon toxin (ETX), produced by , is one of the most potent toxins known, with a lethal potency approaching that of botulinum neurotoxins. Epsilon toxin is responsible for enteritis. Therefore, the development of rapid and simple methods to detect ETX is imperative. Aptamers are single-stranded oligonucleotides that can bind tightly to specific target molecules with an affinity comparable to that of monoclonal antibodies (mAbs). DNA aptamers can serve as tools for the molecular identification of organisms, such as pathogen subspecies.
OBJECTIVES
This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers against ETX.
METHODS
This study identified aptamers using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, enzyme-linked apta-sorbent assay (ELASA), and surface plasmon resonance (SPR) to determine the affinity and specificity of the newly obtained aptamers targeting ETX.
RESULTS
Several aptamers obtained through the SELEX process were studied. Among them, 2 aptamers, ETX clone 3 (ETX3; dissociation constant (Kd = 8.4 ± 2.4E-9M) and ETX11 (Kd = 6.3 ± 1.3E-9M) had favorable specificity for ETX. The limits of detection were 0.21 and 0.08 μg/mL for ETX3 and ETX11, respectively..
CONCLUSIONS
The discovered aptamers can be used in various aptamer-based rapid diagnostic tests for the detection of ETX.
背景
由[未提及的细菌]产生的ε毒素（ETX）是已知最具毒性的毒素之一，其致死效力接近肉毒杆菌神经毒素。ε毒素会引发肠炎。因此，开发快速简便的ETX检测方法势在必行。适体是单链寡核苷酸，能够以与单克隆抗体（mAb）相当的亲和力紧密结合特定靶分子。DNA适体可作为分子鉴定生物的工具，例如病原体亚种。
目的
本研究旨在分离针对ETX的高亲和力单链DNA（ssDNA）适体。
方法
本研究采用指数富集配体系统进化（SELEX）方法、酶联适体吸附测定（ELASA）和表面等离子体共振（SPR）来鉴定适体，以确定新获得的靶向ETX的适体的亲和力和特异性。
结果
研究了通过SELEX过程获得的几种适体。其中，2种适体，即ETX克隆3（ETX3；解离常数（Kd）= 8.4 ± 2.4E - 9M）和ETX11（Kd = 6.3 ± 1.3E - 9M）对ETX具有良好的特异性。ETX3和ETX11的检测限分别为0.21和0.08μg/mL。
结论
发现的适体可用于各种基于适体的快速诊断测试中以检测ETX。
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