基于 ITS2条形码序列对新型“香料”毒品植物成分的研究

摘要

Objective The genetic analysis of ITS2 region presented here is to detect the species of plants from spice drugs using DNA barcoding method. Methods DNA from samples of “spike 99”, “K2” and “7#” collected from cases were extracted using QIAGEN DNeasy plant mini kit. The ITS2 sequence forward primer was 5’-GCGATACTTGGTGTGAAT-3’, and the reverse was 5’-GACGCTTCTCCAGACTACAAT-3’. PCR amplification was performed in a 25µL reaction mixture. Purified PCR products were sequenced in both directions with the primers used for PCR amplification on 3730XL Genetic Analyzer. To estimate the quality of the generated sequence, the original forward and reverse sequences were assembled using CodonCode Aligner V 3.71 and the conserved 5.8S and 26S rRNA sequences were removed from the ITS2 sequence. The PCR amplification products using ITS2 primers were sequenced, analyzed and BLAST in NCBI database. Results The samples of “spike 99”, “K2” and “7#” contained organic compounds which had been determined by GC-MS. “spike 99” was found to possess JWH–073 and JWH–018 components, and JWH–018 in “K2”, cannabis in “7#”, respectively. As for the DNA analysis, one sequence from both “spike 99” and “7#” and two sequences from “K2” were 100% homologous with the ITS2 sequences of Medicago sativa, cannabis sativa, humulus lupulus and Abelmoschus manihot. Conclusions All samples containing constituents from at least four kinds of plants: Medicago sativa, cannabis sativa, humulus lupulus and Abelmoschus manihot. The results were consistent with their chemical constituents and gave the evidence that the DNA approach was an essential alternative to identify the species of plant for the drug abused. DNA barcoding, involving the amplification and sequencing of relatively short, standardized genetic loci, provides a powerful tool to identify the species of plant.%目的：应用 DNA 条形码片段 ITS2对新型毒品样品中的植物成分进行分析。方法提取可疑毒品样本“spike 99”，“K2”，“7号”中的植物基因组 DNA，用 ITS2通用引物进行扩增，对扩增产物进行测序，对序列校对拼接，应用 BLAST 方法在 NCBI 数据库中比对。结果“spike 99”、“7号”各得到1条 ITS2基因序列，“K2”得到2条 ITS2基因序列，分别与紫花苜蓿、大麻、啤酒花、黄蜀葵的 ITS2基因序列的同源性为100%。结论应用条形码技术可以成功分析新型毒品中的植物种属，为案件中植物样本的种属鉴定提供方法。