Rapid identification of Hebeloma crustuliniforme species using real-time fluorescence and visual loop-mediated isothermal amplification based on the internal transcribed spacer sequence

Abstract

Mushroom poisoning is responsible for nearly 50% of all oral poisoning-related deaths in China. Most mushroom poisoning incidences are caused by the difficulty in distinguishing whether wild mushrooms are poisonous or not. In this study, we aimed to develop two rapid and sensitive methods for Hebeloma crustuliniforme identification using visual and real-time loop-mediated isothermal amplification (LAMP) technology. LAMP primers were designed targeting the Hebeloma crustuliniforme internal transcribed spacer (ITS) sequence and their specificity was tested against 41 other mushroom species. We found that the LAMP primers could specifically recognize the Hebeloma crustuliniforme ITS sequence without cross reacting with that of other mushrooms. Both LAMP methods could detect as low as 1 pg/μL genomic DNA and 0.01% Hebeloma crustuliniforme in different mushroom mixtures. The whole detection process was completed within 35 min, with no requirement of complicated instruments for performing visual LAMP. The two methods established in this study can not only be used for the identification of fresh mushrooms but also for cooked mushrooms and mushrooms digested by gastric juice. To the best of our knowledge, this is the first study to establish a detection method for Hebeloma crustuliniforme identification. This rapid, sensitive, and visual method could be used on-site for poisonous mushroom detection and prevention of possible food poisoning incidents.