Abstract
Amanita poisoning is one of the most deadly types of mushroom poisoning. α-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 α-amanitin require expensive instruments. In this study, the α-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 α-amanitin. The detection limit was 0.369 μg/mL, while, in mushroom it was 0.372 μg/mL and in urine 0.337 μg/mL. Recovery studies were performed by spiking urine and mushroom samples with α-amanitin, and these confirmed the desirable accuracy and practical applicability of our method. The α-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 α-amanitin aptamer and proposes a method of detecting α-amanitin.
Highlights
Publisher’s Note: MDPI stays neutralMushroom poisoning is a global problem, which poses a threat to human health
One study reported that 90% of deaths from mushroom poisoning were caused by mushrooms containing amatoxin [2]. α-Amanitin is the most abundant lethal toxin among the amatoxins, with a lethal dose to humans of about 0.1 mg/kg [3]
We developed a magnetic bead-based enzyme linked immunoassay (MELISA) to detect α-amanitin, and this represents the first use of aptamer for aα-amanitin detection
Summary
Mushroom poisoning is a global problem, which poses a threat to human health. Due to accidental picking and consumption [1]. One study reported that 90% of deaths from mushroom poisoning were caused by mushrooms containing amatoxin [2]. Α-Amanitin is the most abundant lethal toxin among the amatoxins, with a lethal dose to humans of about 0.1 mg/kg [3]. When wild mushrooms containing amatoxin are accidentally consumed, the amatoxin enters the hepatocytes and binds to RNA polymerase II, which inhibits the transcription of mRNA and protein synthesis, eventually leading to necrosis of hepatocytes. It has been reported that polymyxin B competes for the RNA polymerase II binding site; inhibiting the binding of α-amanitin to RNA polymerase
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