Manufacturing of a novel double-function ssDNA aptamer for sensitive diagnosis and efficient neutralization of SEA

• Published in: Analytical biochemistry 2018-05, Vol.548, p.69-77
• Main Authors: Sedighian, Hamid, Halabian, Raheleh, Amani, Jafar, Heiat, Mohammad, Taheri, Ramezan Ali, Imani Fooladi, Abbas Ali
• Format: Article
• Language: English
• Subjects: Aptamer shift mobility assay; Aptamers, Nucleotide - chemistry; Enterotoxins - analysis; Enterotoxins - antagonists & inhibitors; Food Analysis - methods; Humans; Neutralization; SELEX Aptamer Technique; ssDNA-aptamer; Staphylococcal enterotoxin A; Surface Plasmon Resonance; Taguchi method
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2018.02.017

Staphylococcal enterotoxin A (SEA) is an enterotoxin produced mainly by Staphylococcus aureus. In recent years, it has become the most prevalent compound for staphylococcal food poisoning (SFP) around the world. In this study, we isolate new dual-function single-stranded DNA (ssDNA) aptamers by using some new methods, such as the Taguchi method, by focusing on the detection and neutralization of SEA enterotoxin in food and clinical samples. For the asymmetric polymerase chain reaction (PCR) optimization of each round of systematic evolution of ligands by exponential enrichment (SELEX), we use Taguchi L9 orthogonal arrays, and the aptamer mobility shift assay (AMSA) is used for initial evaluation of the protein-DNA interactions on the last SELEX round. In our investigation the dissociation constant (KD) value and the limit of detection (LOD) of the candidate aptamer were found to be 8.5 ± 0.91 of nM and 5 ng/ml using surface plasmon resonance (SPR). In the current study, the Taguchi and mobility shift assay methods were innovatively harnessed to improve the selection process and evaluate the protein–aptamer interactions. To the best of our knowledge, this is the first report on employing these two methods in aptamer technology especially against bacterial toxin.