Rapid identification of Colchicum autumnale based on loop-mediated isothermal amplification (LAMP) assay
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Rapid identification of Colchicum autumnale based on loop‑mediated isothermal amplification (LAMP) assay Sae Misawa1 · Daigo Natsuhara2 · Yuka Kiba1 · Tadashi Yamamuro3 · Ryuichiro Suzuki1 · Takayuki Shibata2 · Masashi Kitamura1 Received: 5 August 2020 / Accepted: 18 October 2020 © Japanese Association of Forensic Toxicology 2020
Abstract Purpose Colchicum autumnale (Colchicaceae) exhibits toxicity, and severe poisoning cases due to ingestion of this plant have been reported in Japan. Identifying the cause of poisoning is important for emergency medical treatment, and a rapid and simple detection technique is required for the identification of the cause of poisoning. In the present study, we developed a rapid and simple method for the detection of C. autumnale based on a loop-mediated isothermal amplification (LAMP) assay, which is 100- to 1000-fold higher than the conventional polymerase chain reaction method. Methods Specific LAMP primers for C. autumnale were designed based on the trnL-trnF intergenic spacer region. Using the LAMP primers, the LAMP assay was performed at an isothermal reaction temperature of 63 ℃. Results The LAMP reaction was shown to be specific and highly sensitive to C. autumnale, given that the assay can be used for 10 pg of purified DNA. Using a simple protocol for on-site detection, the entire procedure from pretreatment to evaluation required approximately 1 h. Moreover, the LAMP method using a microfluidic device detected multiple genes, including C. autumnale-specific DNA regions, at a time. Conclusions The currently proposed protocol exhibits good potential as a screening method for C. autumnale poisoning in emergency medical care. Keywords Colchicum · Isothermal DNA amplification · LAMP · Identification of toxic plants · Colchicum autumnale · On-site identification
Introduction In many cases, accidental ingestion of toxic plants occurs because of morphological similarity to edible plants. Colchicum autumnale, which belongs to the Colchicaceae family, originates in North Africa and is cultivated as a garden plant in Japan. The leaves and bulbs of the plant contain colchicine, which is an alkaloid [1]. In Japan, 22 patients were reported to be poisoned by C. autumnale from 2010 to * Masashi Kitamura [email protected] 1
Laboratory of Pharmacognocy, School of Pharmacy, Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, 1–1 Keyakidai, Sakado, Saitama 350‑0295, Japan
2
Department of Mechanical Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441‑8580, Japan
3
National Research Institute of Police Science, 6‑3‑1 Kashiwanoha, Kashiwa, Chiba 277‑0882, Japan
2019, 10 of whom died [2]. In some cases, the leaves of the plant were accidentally consumed as Allium victorialis or Hosta sieboldiana, and the bulbs of the plant were ingested as onions (Allium cepa) or potatoes (Solanum tuberosum). The initial symptoms of C. autumnale poisoning include abdominal pain and vomiting [3–5]. In some serious cases, the toxicity of
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