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Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM)
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 iden...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Taylor & Francis
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9639532/ https://www.ncbi.nlm.nih.gov/pubmed/36353314 http://dx.doi.org/10.1080/20961790.2021.1875580 |
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author | Zhang, Xiaochun Yu, Huan Wang, Ziwei Yang, Qi Xia, Ruocheng Qu, Yiling Tao, Ruiyang Shi, Yan Xiang, Ping Zhang, Suhua Li, Chengtao |
author_facet | Zhang, Xiaochun Yu, Huan Wang, Ziwei Yang, Qi Xia, Ruocheng Qu, Yiling Tao, Ruiyang Shi, Yan Xiang, Ping Zhang, Suhua Li, Chengtao |
author_sort | Zhang, Xiaochun |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9639532 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
spelling | pubmed-96395322022-11-08 Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM) Zhang, Xiaochun Yu, Huan Wang, Ziwei Yang, Qi Xia, Ruocheng Qu, Yiling Tao, Ruiyang Shi, Yan Xiang, Ping Zhang, Suhua Li, Chengtao Forensic Sci Res Regular Papers 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. Taylor & Francis 2021-04-13 /pmc/articles/PMC9639532/ /pubmed/36353314 http://dx.doi.org/10.1080/20961790.2021.1875580 Text en © 2021 The Author(s). Published by Taylor & Francis Group on behalf of the Academy of Forensic Science. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Regular Papers Zhang, Xiaochun Yu, Huan Wang, Ziwei Yang, Qi Xia, Ruocheng Qu, Yiling Tao, Ruiyang Shi, Yan Xiang, Ping Zhang, Suhua Li, Chengtao Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM) |
title | Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM) |
title_full | Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM) |
title_fullStr | Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM) |
title_full_unstemmed | Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM) |
title_short | Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM) |
title_sort | multi-locus identification of psilocybe cubensis by high-resolution melting (hrm) |
topic | Regular Papers |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9639532/ https://www.ncbi.nlm.nih.gov/pubmed/36353314 http://dx.doi.org/10.1080/20961790.2021.1875580 |
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