Novel SCAR markers for accurate diagnosis of toxic Chlorophyllum molybdites and C. globosum

Abstract

Toxic Chlorophyllum molybdites and C. globosum are mistaken for wild edible mushroom species under the same genus, and those under Macrolepiota. These species have similar morphological characteristics and occupy the same ecosystems. Accidental ingestion of C. molybdites and C. globosum can lead to gastrointestinal syndromes including nausea, vomiting, and diarrhea. In severe cases, clinical manifestations, such as dehydration, bloody stools, and profuse sweating, may occur. Given that diagnostic characteristics of poisonous mushrooms are inadequate, the use of molecular techniques is needed. In this study, we developed specific SCAR markers for toxic C. molybdites and C. globosum and used to simultaneously distinguish these toxic species via multiplex PCR. Thirty-five Chlorophyllum and Macrolepiota mushroom samples were obtained, and phylogenetic analysis of the internal transcribed spacer region confirmed their taxonomic entities. High annealing temperature-random amplified polymorphic DNA profiling of all samples using 30 arbitrary primers resulted in nine distinct fragments of C. molybdites and C. globosum, which were further analyzed using the sequence-characterized amplified region (SCAR) technique. Two SCAR primer sets were selected for the accurate discrimination of C. molybdites and C. globosum with expected sizes of 288 and 183 bp, respectively. In the sensitivity tests, both species-specific SCAR markers were detected at DNA template concentration as low as 3 pg/μL in simplex PCR. The presence of these toxic species was detected at 1 % in cooked mushroom samples. Thus, the developed SCAR markers are ideal for the rapid diagnosis of gastrointestinal irritant mushrooms, which may help minimize the risk of severe foodborne illnesses.