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.
Highlights
There are hundreds of hallucinogenic mushrooms found in nature that contain active compounds, and most of them can be abused and hazardous to human health [1].In recent years, the consumption of natural biological products with hallucinogenic properties, such as Psilocybe cubensis, has greatly increased [2,3]
We suggested the high-resolution melting (HRM) assay to evaluate the specificity of internal transcribed spacer (ITS), ITS1, and ITS2 amplicons from 36 mushroom species, including Psilocybe cubensis and other related species
Based on a combination of three Tm values and melting peaks, Psilocybe cubensis could be distinguished from the other species
Summary
There are hundreds of hallucinogenic mushrooms found in nature that contain active compounds, and most of them can be abused and hazardous to human health [1].In recent years, the consumption of natural biological products with hallucinogenic properties, such as Psilocybe cubensis, has greatly increased [2,3]. There are hundreds of hallucinogenic mushrooms found in nature that contain active compounds, and most of them can be abused and hazardous to human health [1]. It is imperative to identify hallucinogenic mushrooms or plants related to criminal proceedings, such as drug-related deaths and drug trafficking, which is of great significance in forensic investigations. The detection of hallucinogenic mushrooms and plants primarily relies on methods ranging from morphological and histological characteristics, gas chromatographymass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and other toxicological examinations [6,7,8]. Several lines of studies have used DNA sequences for inference in cradle and hallucinogenic mushrooms or plants species with a phylogenetic relationship [10,11,12,13]. The DNA barcoding assay is a genetic approach that discriminated one or a few specific DNA regions for species identification
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