Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for ‎Detection of Epsilon Toxin

Abstract

Background: Epsilon toxin (ETX), produced by Clostridium perfringens, 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.