Development of a PCR Assay Targeting the iboH Gene to Assess the Distribution of Ibotenic Acid/Muscimol Biosynthesis Potential in the Genus Amanita

Ibotenic acid and muscimol, synthesized by the ibotenic acid biosynthetic gene cluster (ibo BGC) in Amanita species, are two of the primary neurotoxins responsible for mushroom poisoning incidents worldwide. However, the molecular detection methods for these toxins remain underdeveloped. In this study, we designed a pair of universal PCR primers targeting iboH, a hydroxylase-encoding gene within the ibo BGC, and developed a PCR assay with a detection limit of 10 pg of genomic DNA per reaction. Using the assay on 53 Amanita species, 21 out of the 23 species tested from section Amanita were positive, whereas all 30 species from sects. Amidella (1), Caesareae (3), Phalloideae (6), Roanokenses (8), Strobiliformes (1), Vaginatae (4), and Validae (7) tested negative. We then integrated these PCR signals into our global phylogenetic analysis of Amanita and found that iboH is restricted to a large clade within sect. Amanita. Amanita sinensis, commonly considered an edible species in China, was detected carrying iboH, indicating its potential to produce ibotenic acid/muscimol and pose associated health risks. In conclusion, this study establishes an effective universal PCR assay for detecting ibotenic acid/muscimol-producing Amanita species and offer a reference for toxigenic mushroom recognition and food safety.