Rapid Identification of Amanita Citrinoannulata Poisoning Using Colorimetric and Real-Time Fluorescence and Loop-Mediated Isothermal Amplification (LAMP) Based on the Nuclear its Region

Abstract

The health concerns and financial losses caused by mushroom poisoning have been reported worldwide. Amanita citrinoannulata, a poisonous mushroom commonly found in China, results in a toxic reaction in some people after accidental ingestion. Thus, the establishment of a rapid identification method for mushroom poisoning sources for correct patient treatment is of great significance. Further, such identification methods will be advantageous in the identification of other poisonous mushroom species. This study establishes two rapid and sensitive methods for the detection of Amanita citrinoannulata. The methods use colorimetric and real-time loop-mediated isothermal amplification (LAMP) technology and specifically designed primers for the internal transcribed spacer (ITS) genes of A. citrinoannulata. The methods demonstrated high sensitivity as 0.2 ng of A. citrinoannulata DNA could be detected, with no cross-reaction with 41 non-target mushroom species. The entire detection process could be completed within 40 min without requiring complex instruments and can be observed by the naked-eye. Therefore, the novel method can be used for the identification of fresh and cooked mushroom samples, as well as vomit samples, which contain only 1% A. citrinoannulata. This novel method enables the detection of mushroom poisoning and, thus, has potential to reduce the number of mushroom poisoning-related deaths worldwide.

Keywords: ITS region, Loop-mediated isothermal amplification (LAMP), authentication, Amanita citrinoannulata