Utilizing the DNA Aptamer to Determine Lethal alpha-Amanitin in Mushroom Samples and Urine by Magnetic Bead-ELISA (MELISA)

Amanita poisoning is one of the most deadly types of mushroom poisoning. alpha-Amanitin is the main lethal toxin in amanita, and the human-lethal dose is about 0.1 mg/kg. Most of the commonly used detection techniques for alpha-amanitin require expensive instruments. In this study, the alpha-amanitin aptamer was selected as the research object, and the stem-loop structure of the original aptamer was not damaged by truncating the redundant bases, in order to improve the affinity and specificity of the aptamer. The specificity and affinity of the truncated aptamers were determined using isothermal titration calorimetry (ITC) and gold nanoparticles (AuNPs), and the affinity and specificity of the aptamers decreased after truncation. Therefore, the original aptamer was selected to establish a simple and specific magnetic bead-based enzyme linked immunoassay (MELISA) method for alpha-amanitin. The detection limit was 0.369 mu g/mL, while, in mushroom it was 0.372 mu g/mL and in urine 0.337 mu g/mL. Recovery studies were performed by spiking urine and mushroom samples with alpha-amanitin, and these confirmed the desirable accuracy and practical applicability of our method. The alpha-amanitin and aptamer recognition sites and binding pockets were investigated in an in vitro molecular docking environment, and the main binding bases of both were T3, G4, C5, T6, T7, C67, and A68. This study truncated the alpha-amanitin aptamer and proposes a method of detecting alpha-amanitin.