Development of a DNA-Based Macroarray for the Detection and Identification of Amanita Species

Abstract

A DNA-based macroarray was designed to quickly and accurately identify certain Amanita mushroom specimens at the species level. The macroarray included probes for Amanita phalloides and Amanita ocreata, toxic species responsible for most mushroom poisonings, and Amanita lanei and Amanita velosa, edible species sometimes confused with toxic species, based on sequences of the highly variable internal transcribed spacer (ITS) region of rDNA. A cryptic species related to A. ocreata and one related to A. lanei, identifiable by ITS sequences, were also included. Specific multiple oligonucleotide probes were spotted onto nylon membranes and the optimal hybridization temperatures were determined. The Amanita DNA array was highly specific, sensitive (0.5 ng DNA/μL and higher were detected), and reproducible. In two case studies, the method proved useful when only small amounts of mushroom tissue remained after a suspected poisoning. An identification could be completed in 12 h.