Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Abstract

Psilocybe cubensis, or "magic mushroom," is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web-based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR Green I, Radiant™ Green, or LCGreen Plus intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant™ Green dye.

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

“…The HRM melting curve has prominent capacity to discriminate DNA barcodes based on their composition, length, or GC content, which allows for the examination of genetic variations within DNA sequences. Moreover, it supports forensic evidence of the suspicious species [19][20][21][22][23]. For example, it has been demonstrated that HRM could identify Cannabis sativa and "legal high" drugs, among other species [21].…”

“…For example, it has been demonstrated that HRM could identify Cannabis sativa and "legal high" drugs, among other species [21]. A pioneer study also proposed a PCR HRM duplex assay that can simultaneously detect Psilocybe cubensis and Cannabis sativa DNA [23]. In comparison to other discriminatory methods, the HRM analysis is a rapid, user-friendly, cost-effective, and highly reproducible approach that can accurately screen a large number of samples for the presence of specific targets, including the variations within the ITS region.…”

High‐resolution melting of the cytochrome B gene in fecal DNA: A powerful approach for fox species identification of the Lycalopex genus in Chile

“…The reaction mixture for real‐time PCR and HRM analysis consisted of a total volume of 15 µl. This contained 7.5 µl of 2× Kapa SYBR qPCR Mix (Kapa Biosystems Roche), (Cowan & Elkins, ; Solano et al, ) 0.2 µM forward primer, 0.2 µM reverse primer, and 1 µl of 25 ng/µl DNA. SYBR fluorescence dye was used to monitor the accumulation of the amplified product during PCR and the HRM process to derive the T m value.…”

Interpol review of controlled substances 2016–2019

“…Mushrooms (including Psilocybe mushrooms): 2016 MC-ICPMS method for precise V-51/V-50 isotope ratio measurements as a useful tool for identifying the origin of Amanita muscaria - a widespread toxic and hallucinogenic mushroom [ 783 ]; identification of mushroom samples from nine clinically reported cases in Thailand during a 7-year period based on nuclear ITS sequence data and identification of lethal peptide toxins using a reversed phase LC-MS method [ 784 ]; DNA-based taxonomic identification of basidiospores in hallucinogenic mushrooms cultivated in “grow-kits” seized by the police: LC-UV quali-quantitative determination of psilocybin and psilocin [ 785 ]; 2017 i dentification and quantitation of Psilocybe cubensis DNA using a quantitative qPCR-HRM assay [ 786 ]; review [ 787 ]; characterization of four psilocybin biosynthesis enzymes [ 788 ]; 1D and 2D NMR spectroscopy and high-resolution mass spectrometry for the identification of norpsilocin in Psilocybe cubensis [ 789 ]; isolation of 10 compounds from Psilocybe merdaria and structural determination from the analysis of 1D and 2D NMR and MS data [ 790 ]; characterization of four psilocybin biosynthesis enzymes [ 788 ]; 2018 Detection and identification of psilocybe cubensis DNA using a real-time PCR assay [ 791 ]; an enhanced enzymatic route of psilocybin production by adding the tryptophan synthase of the mushroom Psilocybe cubensis (TrpB) to the reaction [ 792 ]; review [ 793 ]; 2019 review [ 794 ]; morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory using SEM, MS, HRM and ITS sequencing [ 795 ];…”