Macrofungal Diversity of North East India and Development of Nanoparticle Based Detection of Mushroom Toxin

Abstract
Mushrooms have been an important part of the diet for the people worldwide due to its nutritious and delectable taste since ancient times. The ethnic communities of the Northeast India have extensive traditional knowledge of the edible mushrooms which they forage from the wilds. Unfortunately, increasing population pressure and consumer demand for exotic mushrooms have led to indiscriminate collection and sale of unidentified varieties leading to lethal cases on several occasion. Development of diagnostic kits to detect toxins present in wild mushrooms in situ for prevention and detection of mushroom poisoning is therefore very important and can aid in rapid detection of the toxins in affected patients for early treatment. A systematic collection of mushrooms and their detailed study of diversity and distribution of wild mushrooms in the state of Assam based on phenotypic and molecular characteristics led to a collection of 44 samples out of which, 17 mushrooms species were found predominantly during summer season, 11 species during autumn, nine species during monsoon, three species in winter and eight species during spring season. Soil was preferred habitat followed by tree/hardwood tree. Molecular characterization based on rDNA-ITS sequences revealed 16 macrofungal families. Out of the 44 samples, 23 samples were reported to be edible and for the other 21 non-edible strains, five strains had medicinal properties, six strains were earlier reported poisonous, two had industrial application while the properties of the rest are yet to be ascertained. Phallacidin, a bicyclic heptapeptide of the phallotoxin family is highly hepatotoxic and found in many of the poisonous mushrooms. This study generated antibodies against the Phallacidin (PCN) toxin present in poisonous mushrooms using Phallacidin-BSA conjugate in New Zealand white rabbit. The antibodies showed high sensitivity and detection limit of 11.89 ng/mL for phallacidin and 8.6 ng/mL for α-Amanitin. The detection limits with reduced assay time for these two toxins were further improved to 10.87 ng/mL (Phallacidin) and 11.09 ng/mL (α-Amanitin) through generation of HRP-PCN conjugate. A lateral-flow-based dipstick immunoassay format using antibody-gold conjugate for rapid field screening of Phallacidin with a detection limit of 25 ng/mL was further developed. The present study reports development of three methods viz. ELISA, HRP-PCN and lateral flow immunoassay for detection of Phallacidin & α-Amanitin. This is the first report of development of immunoassay to detect Phallacidin toxin. The immunoassays developed through this study can be convenient quantitative tool for screening of toxin in wild mushrooms.
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