Rapid identification of Gloriosa superba and Colchicum autumnale by melting curve analysis: application to a suicide case involving massive ingestion of G. superba

• Published in: International journal of legal medicine Vol. 133; no. 4; pp. 1065 - 1073
• Main Authors: Sakurada, Makoto, Yoshioka, Naoki, Kuse, Azumi, Nakagawa, Kanako, Morichika, Mai, Takahashi, Motonori, Kondo, Takeshi, Asano, Migiwa, Ueno, Yasuhiro
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.07.2019; Springer Nature B.V
• Subjects: Colchicine; Colchicine - poisoning; Deoxyribonucleic acid; DNA; Drug Overdose - diagnosis; Emergency medical services; Flowers & plants; Forensic Medicine; Forensic pathology; Forensic toxicology; Humans; Ingestion; Medical Law; Medicine; Medicine & Public Health; Melting; Original Article; Plants, Toxic - poisoning; Real time; Real-Time Polymerase Chain Reaction - methods; Stomach; Suicide; Suicides & suicide attempts; Toxicity; Melting curve analysis; Forensic analysis of stomach contents; Colchicine; Real-time PCR; Gloriosa superba; Colchicum autumnale
• ISSN: 0937-9827; 1437-1596
• DOI: 10.1007/s00414-019-02060-x

The plant species Gloriosa superba and Colchicum autumnale produce extremely poisonous colchicine as a major toxic metabolite. Almost all previous studies on colchicine poisoning have focused on drug analysis and clinical and pathological aspects. In this study, we developed a rapid, highly sensitive method to identify G. superba and C. autumnale. This method, which can distinguish between G. superba and C. autumnale using even minute amounts of plant material, is based on duplex real-time PCR in combination with melting curve analysis. To discriminate between the two genera of colchicine-containing plants, we designed new primer pairs targeting the region of the ycf15 gene, which is present in C. autumnale but not G. superba . By producing PCR amplicons with easily distinguishable melting temperatures, we were able to rapidly and accurately distinguish G. superba from C. autumnale . The new primer pairs generated no PCR amplicons from commercially available human DNA or various plant DNAs except for G. superba and C. autumnale . Sensitivity testing indicated that this assay can accurately detect less than 0.031 ng of DNA. Using our method in conjunction with colchicine drug analysis, we successfully identified G. superba in the stomach contents of a suicide victim who ingested massive quantities of a colchicine-containing plant. According to these results, duplex real-time PCR analysis is very appropriate for testing forensic samples, such as stomach contents harboring a variety of vegetables, and enables discrimination between G. superba and C. autumnale in forensic and emergency medical fields.