Hwang Eun-Mi, Jeong Kyu-Sik, Yoo Seong Yeon, Kim Jihyun, Choe Sanggil, Kim Joo-Young
Forensic DNA Division, National Forensic Service, Wonju, 26460, Republic of Korea.
Forensic Toxicology and Chemistry Division, Seoul Institute, National Forensic Service, Seoul, 08036, Republic of Korea.
Int J Legal Med. 2025 Jan;139(1):1-13. doi: 10.1007/s00414-024-03318-9. Epub 2024 Aug 27.
The Lophophora genus of the Cactaceae family includes Lophophora diffusa and Lophophora williamsii, which has traditionally been used as a natural analgesic; however, its use is now under strict regulation worldwide as it contains mescaline, a unique psychotropic agent. Recently, non-medical and illegal distribution and abuse of L. williamsii have increased worldwide; thus, effective species identification methods are urgently needed. Here, we identified a new variable number tandem repeat (VNTR) marker in the trnL intron region to identify and characterize species in forensic analyses. The VNTR marker has a unique structure of tandem repeats, each with 13 nucleotides; one repeat unit was found in L. williamsii and two in L. diffusa. Phylogenetic and length polymorphism analyses confirmed that this novel VNTR marker could distinguish between Lophophora species. Furthermore, our newly developed TaqMan genotyping assay utilizes two probes; the color and position of dots on the discrimination plot differ according to the tandem repeat count within the VNTR marker. The limits of detection of the assay were 0.000063 ng (LW-VNTR probe-1) and 0.000066 ng (LW-VNTR probe-2), indicating high sensitivity. Moreover, when crime scene samples of 16 presumed L. williamsii species were analyzed, the results coincided with those of gas chromatography-mass spectrometry, confirming the applicability of our marker for Lophophora species identification. Thus, the tandem repeats within the trnL intron region can be exploited as a VNTR marker to identify L. williamsii and L. diffusa. Our dual TaqMan genotyping assay based on a novel marker demonstrates potential for forensic applications.
仙人掌科的乌羽玉属包括散鳞乌羽玉和威廉斯氏乌羽玉，传统上被用作天然止痛剂；然而，由于其含有三甲氧苯乙胺这种独特的精神药物，目前其使用在全球范围内受到严格监管。最近，威廉斯氏乌羽玉在全球范围内的非医疗和非法分销及滥用情况有所增加；因此，迫切需要有效的物种鉴定方法。在此，我们在trnL内含子区域鉴定出一个新的可变数目串联重复（VNTR）标记，用于法医分析中的物种鉴定和特征描述。该VNTR标记具有独特的串联重复结构，每个重复单元有13个核苷酸；在威廉斯氏乌羽玉中发现一个重复单元，在散鳞乌羽玉中发现两个重复单元。系统发育和长度多态性分析证实，这个新的VNTR标记可以区分乌羽玉属的不同物种。此外，我们新开发的TaqMan基因分型检测方法使用了两种探针；根据VNTR标记内串联重复的数量，判别图上点的颜色和位置会有所不同。该检测方法的检测限分别为0.000063 ng（LW-VNTR探针-1）和0.000066 ng（LW-VNTR探针-2），表明灵敏度很高。此外，当对16个疑似威廉斯氏乌羽玉物种的犯罪现场样本进行分析时，结果与气相色谱-质谱分析结果一致，证实了我们的标记物在乌羽玉属物种鉴定中的适用性。因此，trnL内含子区域内的串联重复可以作为VNTR标记来鉴定威廉斯氏乌羽玉和散鳞乌羽玉。我们基于新标记物的双重TaqMan基因分型检测方法显示出在法医应用中的潜力。
