Department of Forensic Science, Medical School of Soochow University, Suzhou 215123, China.
Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Science, Ministry of Justice, Shanghai 200063, China.
Genes (Basel). 2021 Jan 29;12(2):199. doi: 10.3390/genes12020199.
In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of and other fungi, which can be utilized for drug trafficking cases and forensic science.
近年来，迷幻蘑菇的贩运和滥用已成为一个严重的社会问题。因此，迫切需要为国家毒品管制立法确定致幻蘑菇属。内部转录间隔区 (ITS) 是一种用于物种鉴定的 DNA 条形码工具。已经使用了许多方法来区分 ITS 区域，但它们通常受到分辨率低的限制。在这项研究中，我们试图通过高分辨率熔解 (HRM) 分析来分析 ITS 及其片段 ITS1 和 ITS2，HRM 分析是一种快速而敏感的方法，用于评估 PCR 扩增子内的序列变异。对 ITS HRM 检测进行了特异性、重现性、灵敏度和分析混合物样品的能力测试。结果表明，的 ITS、ITS1 和 ITS2 的熔点分别为 83.72±0.01°C、80.98±0.06°C 和 83.46±0.08°C，对于其他物种，我们也得到了物种特异性的结果。最后，我们进行了 ITS 测序以验证我们样本的假定分类学身份，测序结果显著支持了我们的 HRM 数据。综上所述，这些结果表明 HRM 方法可以快速区分致幻蘑菇属和其他真菌的 DNA 条形码，可用于毒品贩运案件和法医学。
