致幻蘑菇是一种含有精神活性色胺物质如psilocybin的有毒菌株,psilocin和ibotenic酸,等。含有致幻成分的蘑菇种类繁多,分布广泛,缺乏标准来定义,这对身份识别提出了巨大挑战。传统的识别方法,如形态学和毒理学分析,在旧样品或加工样品中显示出缺陷,而基于DNA的致幻蘑菇鉴定将允许鉴定这些样品,由于DNA的稳定性。在本文中,设计了四个引物组以PsillocybecubensisDNA为目标,以提高本鉴定方法的分辨率,目标标记包括RNA聚合酶II的最大亚基(标记为PC-R1),psilocybin相关磷酸转移酶基因(标记为PC-PT),甘油醛3-磷酸脱氢酶(标记为PC-3)和翻译EF1α(标记为PC-EF)。具有高分辨率解链(HRM)测定的实时PCR用于区分由这些引物组扩增的片段,进行了特异性测试,再现性,灵敏度,混合物分析和多重PCR。结果表明,PC-R1,PC-PT,猪猪的PC-3和PC-EF为(87.93±0.12)℃,(82.21±0.14)°C,在我们的独立实验中,分别为(79.72±0.12)°C和(80.11±0.19)°C。用低浓度的62.5pg/µLDNA样品可以显示出显著的HRM特性,并且可以在与智人或大麻的混合物中检测到。总之,HRM分析的方法可以快速而具体地区别于其他物种。可以用于法医学,医疗诊断和贩毒案件。本文的补充数据可在https://doi.org/10.1080/20961790.202.1875580在线获得。
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.