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...
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2021-01-01
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Online Access: | http://dx.doi.org/10.1080/20961790.2021.1875580 |
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doaj-f759b022f4414b2c96c5459a673cb6422021-04-21T16:14:25ZengTaylor & Francis GroupForensic Sciences Research2096-17902471-14112021-01-01001810.1080/20961790.2021.18755801875580Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM)Xiaochun Zhang0Huan Yu1Ziwei Wang2Qi Yang3Ruocheng Xia4Yiling Qu5Ruiyang Tao6Yan Shi7Ping Xiang8Suhua Zhang9Chengtao Li10Department of Forensic Science, Medical School of Soochow UniversityDepartment of Forensic Science, Medical School of Soochow UniversityDepartment of Forensic Science, Medical School of Soochow UniversityDepartment of Forensic Science, Medical School of Soochow UniversityShanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of JusticeDepartment of Forensic Science, Medical School of Soochow UniversityShanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of JusticeShanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of JusticeShanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of JusticeShanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of JusticeDepartment of Forensic Science, Medical School of Soochow UniversityHallucinogenic 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 is available online at https://doi.org/10.1080/20961790.2021.1875580.http://dx.doi.org/10.1080/20961790.2021.1875580forensic sciencesforensic geneticspolymerase chain reaction (pcr)high-resolution melting (hrm) psilocybe cubensis multi-locusspecies-specific |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Xiaochun Zhang Huan Yu Ziwei Wang Qi Yang Ruocheng Xia Yiling Qu Ruiyang Tao Yan Shi Ping Xiang Suhua Zhang Chengtao Li |
spellingShingle |
Xiaochun Zhang Huan Yu Ziwei Wang Qi Yang Ruocheng Xia Yiling Qu Ruiyang Tao Yan Shi Ping Xiang Suhua Zhang Chengtao Li Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM) Forensic Sciences Research forensic sciences forensic genetics polymerase chain reaction (pcr) high-resolution melting (hrm) psilocybe cubensis multi-locus species-specific |
author_facet |
Xiaochun Zhang Huan Yu Ziwei Wang Qi Yang Ruocheng Xia Yiling Qu Ruiyang Tao Yan Shi Ping Xiang Suhua Zhang Chengtao Li |
author_sort |
Xiaochun Zhang |
title |
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_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_sort |
multi-locus identification of psilocybe cubensis by high-resolution melting (hrm) |
publisher |
Taylor & Francis Group |
series |
Forensic Sciences Research |
issn |
2096-1790 2471-1411 |
publishDate |
2021-01-01 |
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 is available online at https://doi.org/10.1080/20961790.2021.1875580. |
topic |
forensic sciences forensic genetics polymerase chain reaction (pcr) high-resolution melting (hrm) psilocybe cubensis multi-locus species-specific |
url |
http://dx.doi.org/10.1080/20961790.2021.1875580 |
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