With the continued emergence and spread of amatoxin-containing mushrooms globally, there is need for a rapid, accurate, and easy-to-use technology to identify deadly poisonous mushrooms and to improve clinical diagnosis of mushroom poisonings in humans as well as in animals. Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. The mushroom poison that causes the most deaths is amanitin (or amatoxins). Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to high cross-reactivity with other chemicals found in mushrooms.<br/><br/>In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. We tested mushrooms (dried and fresh), as well as urine (human and dog) samples by LFIA. Mushrooms were extracted in a saline buffer in &lt;1 min and a small aliquot of the sample was applied to the LFIA test strip. Human urine samples were fortified to recapitulate previously identified amatoxins concentrations from an exposure study. Dog urine samples were obtained from suspected mushroom intoxications as well as control samples from healthy and sick animals. Urine samples were directly applied to the LFIA test strip without sample preparation. The LFIA accurately identified 6 mushroom species that were known to contain amatoxins. Analysis of both fortified human urine samples and urine samples from intoxicated dog, using the LFIA test correlated well with liquid-chromatography mass spectrometry (LC-MS) methods. The efficiency of the LFIA in identifying amatoxin positive urine samples ranged from 78.9-94.6% with limited sample sizes (n=38 and n=96, respectively).<br/><br/>The data illustrates excellent analytical performance of the LFIA for the rapid, sensitive, selective, and accurate detection of amatoxins. This assay can be used to address the shortcomings (speed, portability, cost) of current clinical diagnostics, holding high promise to identify fatal mushroom poisonings sooner. In addition, this LFIA can be used to aid mycologists and clinicians to quickly identify amatoxin-containing mushrooms.

Rapid, portable detection of amatoxins (amanitins) by lateral flow immunoassay: Applications using mushrooms and urine samples

ACS Fall 2020 Virtual Meeting

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