Cowan Ashley F, Elkins Kelly M
Chemistry Department, Forensic Science Program, Towson University, 8000 York Rd, Towson, MD, 21252.
J Forensic Sci. 2018 Sep;63(5):1500-1505. doi: 10.1111/1556-4029.13714. Epub 2017 Dec 1.
Psilocybe cubensis, or "magic mushroom," is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web-based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR Green I, Radiant™ Green, or LCGreen Plus intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant™ Green dye.
古巴裸盖菇，即“神奇蘑菇”，是最常见的具有致幻特性的真菌物种。使用基于网络的软件和美国国立生物技术信息中心（NCBI）的基因序列设计了两套引物，用于靶向古巴裸盖菇的DNA。使用Qiagen DNeasy植物微量提取试剂盒从18个样本中提取DNA，其中包括12种蘑菇。使用引物和含有SYBR Green I、Radiant™ Green或LCGreen Plus嵌入染料的预混液通过聚合酶链反应（PCR）扩增DNA；使用琼脂糖凝胶电泳确定扩增子大小。使用靶向大麻的引物对PCR检测进行扩增能力、特异性、可重复性、稳健性、敏感性和多重性测试。使用SYBR Green I染料时，引物组1和7观察到的高分辨率熔解（HRM）温度分别为78.85±0.31°C和73.22±0.61°C，使用Radiant™ Green染料时分别为81.67±0.06°C和76.04±0.11°C。
