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139 Int'l J. Legal Med. 1 (2025)

handle is hein.journals/injlegame139 and id is 1 raw text is: International Journal of Legal Medicine (2025) 139:1-13 https://doi.org/l0.1007/s00414-024-03318-9 ORIGINAL ARTICLE Development of a diagnostic variable number tandem repeat marker and dual TaqMan genotyping assay to distinguish Lophophora species Eun-Mi Hwang' • Kyu-Sik Jeong' • Seong Yeon Yool • Jihyun Kim2 - Sanggil Choe3 - Joo-Young Kim' Received: 16 January 2024 / Accepted: 20 August 2024 / Published online: 27 August 2024 ©The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2024 Abstract The Lophophora genus of the Cactaceae family includes Lophophora diffusa and Lophophora williamsii, which has tradi- tionally 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 world- wide; 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 detec- tion 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 identifica- tion. 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. Keywords Lophophora williamsii - Lophophora diffusa - Mescaline - Variable number tandem repeats - TaqMan genotyping assay - Locked nucleic acid Introduction Peyote is a common name given to the plant Lophophora williamsii of the Cactaceae family. L. williamsii is a small, spineless, greenish-blue succulent, and its native habitat is North America, mainly the alpine regions in Mexico and dry lands in the Southwestern USA. The Lophophora genus includes two species: L. williamsii and Lophophora diffusa [1, 2]. L. williamsii contains mescaline, a psychotropic alka- loid that acts on the central nervous system. The mechanism Joo-Young Kim jykim7112@korea.kr Forensic DNA Division, National Forensic Service, Wonju 26460, Republic of Korea 2 Forensic Toxicology and Chemistry Division, Seoul Institute, National Forensic Service, Seoul 08036, Republic of Korea 3 Forensic Toxicology Division, National Forensic Service, Wonju 26460, Republic of Korea of action of mescaline is similar to that of lysergic acid die- thyl amide (LSD) [3, 4]. As a result, L. williamsii has been designated and controlled as a Schedule 1 Drug, based on the narcotics criteria of the Controlled Substances Act in the USA, since 1970. The use and handling of the plant have also been banned internationally since 1971 according to the Agreement on Psychotropic Substances [1]. In the Republic of Korea, L. williamsii has been designated and strictly regulated as a psychotropic drug according to Item A of Paragraph 3 in Article 2 of the Narcotics Control Act [5]. Thus, accurately identifying L. williamsii is essential for the protection of public health and safety. The conventional methods of Lophophora species iden- tification are morphological classification and component analysis using gas chromatography (GC) or high-perfor- mance liquid chromatography (HPLC) with mass spectrom- etry (MS) to determine mescaline content. However, mesca- line content may vary according to the site of L. williamsii sample collection (bud, stem, root, etc.), and more mescaline '_i Springer