Toxic and Hallucinogenic Mushroom Identification via DNA-based Macroarray

Two DNA-based macroarrays were designed to quickly and accurately identify mushroom specimens at the species level for both toxic and hallucinogenic mushroom species. DNA-based methods can be used as a means to identify mushrooms that cannot be identified by morphological characteristics alone and are likely to become an important tool to identify toxic mushrooms when the mushrooms are prepared for food. A DNA array for species of Amanita was designed based on sequences of the highly variable internal transcribed spacer (ITS) region of nuclear rDNA. The array was designed to detect five species of Amanita, including the deadly Amanita phalloides and A. ocreata and three edible species, A. lanei, A. aff. lanei, and A. velosa. The three edible species are collected for the table and may be easily confused with toxic species. Species-specific, 17--27 base oligonucleotide probes with melting temperatures ranging between 50°C and 60°C were spotted in quadruplicate on nylon membranes. PCR-amplified ITS rDNA from mushroom specimens was labeled and hybridized with complimentary species-specific probes bound to the membranes. Species-level identification was achieved by the application of a detection reagent to the nylon membranes, which catalyzed a light emitting reaction where the labeled probes bound to the membrane. This chemiluminescent reaction was visualized by film exposure, and the pattern of hybridization on each membrane was read using the corresponding membrane key. The array was highly specific, sensitive (0.5 ng DNA/microl and higher were detected), reproducible, and resulted in accurate mushroom identification. Another DNA array was developed to identify the hallucinogenic Psilocybe cubensis for toxicological or legal purposes. In addition to developing the two macroarrays, various regions of the genome of 14 species of Amanita were sequenced to explore phylogenetic relationships.