Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM)

Xiaochun Zhang; Huan Yu; Ziwei Wang; Qi Yang; Ruocheng Xia; Yiling Qu; Ruiyang Tao; Yan Shi; Ping Xiang; Suhua Zhang; Chengtao Li

doi:10.1080/20961790.2021.1875580

Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM)

Forensic Sci Res. 2021 Apr 13;7(3):490-497. doi: 10.1080/20961790.2021.1875580. eCollection 2022.

Authors

Xiaochun Zhang^{1

2}, Huan Yu^{1

2}, Ziwei Wang^{1

2}, Qi Yang^{1

2}, Ruocheng Xia^{2

3}, Yiling Qu^{1

2}, Ruiyang Tao^{2

4}, Yan Shi², Ping Xiang², Suhua Zhang², Chengtao Li^{1

2}

Affiliations

¹ Department of Forensic Science, Medical School of Soochow University, Suzhou, China.
² Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of Justice, Shanghai, China.
³ Department of Forensic Medicine, School of Basic Medical Science, Wenzhou Medical University, Wenzhou, China.
⁴ Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, China.

Abstract

Hallucinogenic mushroom is a kind of toxic strain containing psychoactive tryptamine substances such as psilocybin, psilocin and ibotenic acid, etc. The mushrooms containing hallucinogenic components are various, widely distributed and lack of standard to define, which made a great challenge to identification. Traditional identification methods, such as morphology and toxicology analysis, showed shortcomings in old or processed samples, while the DNA-based identification of hallucinogenic mushrooms would allow to identify these samples due to the stability of DNA. In this paper, four primer sets are designed to target Psilocybe cubensis DNA for increasing resolution of present identification method, and the target markers include largest subunit of RNA polymerase II (marked as PC-R1), psilocybin-related phosphotransferase gene (marked as PC-PT), glyceraldehyde 3-phosphate dehydrogenase (marked as PC-3) and translation EF1α (marked as PC-EF). Real-time PCR with high-resolution melting (HRM) assay were used for the differentiation of the fragments amplified by these primer sets, which were tested for specificity, reproducibility, sensitivity, mixture analysis and multiplex PCR. It was shown that the melting temperatures of PC-R1, PC-PT, PC-3 and PC-EF of P. cubensis were (87.93 ± 0.12) °C, (82.21 ± 0.14) °C, (79.72 ± 0.12) °C and (80.11 ± 0.19) °C in our kinds of independent experiments. Significant HRM characteristic can be shown with a low concentration of 62.5 pg/µL DNA sample, and P. cubensis could be detected in mixtures with Homo sapiens or Cannabis sativa. In summary, the method of HRM analysis can quickly and specifically distinguish P. cubensis from other species, which could be utilized for forensic science, medical diagnosis and drug trafficking cases. Supplemental data for this article are available online at https://doi.org/10.1080/20961790.2021.1875580.

Keywords: Forensic sciences; Psilocybe cubensis; forensic genetics; high-resolution melting (HRM); multi-locus; polymerase chain reaction (PCR); species-specific.