A Forensic Detection Method for Hallucinogenic Mushrooms via High-Resolution Melting (HRM) Analysis

Abstract

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

Interpol Review of Drug Analysis 2019-2022

“…2019 review [ 435 ]; morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory using scanning electron microscopy (SEM), mass spectrometry, HRM analysis, and internal transcribed spacer (ITS) sequencing [ 436 ]; 2020 use of TLC, FTIR and GC-MS to differentiate Psilocin (4-hydroxy-N,N-dimethyltryptamine, 4-HO-DMT) and bufotenine (5-hydroxy-N,N-dimethyltryptamine, 5-HO-DMT) and benzene ring regioisomers, 6-hydroxy-N,N-dimethyltryptamine (6-HO-DMT) and 7-hydroxy-N,N-dimethyltryptamine (7-HO-DMT) [ 437 ]; immunochemical detection method for monitoring psilocybin and psilocin in dried powder of hallucinogenic mushroom (Psilocybe cubensis) [ 438 ]; 2021 chemoenzymatic synthesis of 5-methylpsilocin, a novel analogue of psilocybin with potential psychedelic activity [ 439 ]; stability study of psilocybin and its four analogues (psilocin, baeocystin, norbaeocystin, and aeruginascin) in psilocybe cubensis mushrooms [ 440 ]; medicinal properties and bioactive compounds from 79 species of wild mushrooms native to North America [ 441 ]; DNA-based identification of hallucinogenic mushrooms [ 442 ]; forensic detection method for hallucinogenic mushrooms via High-Resolution Melting (HRM) analysis [ 443 ]; 2022 phytochemicals and antioxidant activity of seven wild mushrooms [ 444 ].…”