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

Abstract

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.

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References

  1. 1.

    Beyer J, Drummer OH, Maurer HH (2009) Analysis of toxic alkaloids in body samples. Forensic Sci Int 185:1–9. https://doi.org/10.1016/j.forsciint.2008.12.006

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Mezzasalma V, Ganopoulos I, Galimberti A, Cornara L, Ferri E, Labra M (2017) Poisonous or non-poisonous plants? DNA-based tools and applications for accurate identification. Int J Legal Med 131:1–19. https://doi.org/10.1007/s00414-016-1460-y

    Article  PubMed  Google Scholar 

  3. 3.

    Ono T, Hayashida M, Uekusa K, Lai CF, Hayakawa H, Nihira M, Ohno Y (2009) An accidental case of aconite poisoning due to Kampo herbal medicine ingestion. Leg Med (Tokyo) 11:132–135. https://doi.org/10.1016/j.legalmed.2008.11.001

    Article  Google Scholar 

  4. 4.

    Schep LJ, Slaughter RJ, Beasley DM (2009) Nicotinic plant poisoning. Clin Toxicol (Phila) 47:771–781. https://doi.org/10.1080/15563650903252186

    CAS  Article  Google Scholar 

  5. 5.

    Jain R, Sharma A, Gupta S, Sarethy IP, Gabrani R (2011) Solanum nigrum: current perspectives on therapeutic properties. Altern Med Rev 16:78–85

    PubMed  Google Scholar 

  6. 6.

    Gilotta I, Brvar M (2010) Accidental poisoning with Veratrum album mistaken for wild garlic (Allium ursinum). Clin Toxicol (Phila) 48:949–952. https://doi.org/10.3109/15563650.2010.533675

    Article  Google Scholar 

  7. 7.

    Alexandre J, Foucault A, Coutance G, Scanu P, Milliez P (2012) Digitalis intoxication induced by an acute accidental poisoning by lily of the valley. Circulation 125:1053–1055. https://doi.org/10.1161/circulationaha.111.044628

    Article  PubMed  Google Scholar 

  8. 8.

    Berdai MA, Labib S, Chetouani K, Harandou M (2012) Atropa belladonna intoxication: a case report. Pan Afr Med J 11:72

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Angelidis C, Kotsialou Z, Kossyvakis C, Vrettou AR, Zacharoulis A, Kolokathis F, Kekeris V, Giannopoulos G (2018) Colchicine pharmacokinetics and mechanism of action. Curr Pharm Des 24:659–663. https://doi.org/10.2174/1381612824666180123110042

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Dalbeth N, Lauterio TJ, Wolfe HR (2014) Mechanism of action of colchicine in the treatment of gout. Clin Ther 36:1465–1479. https://doi.org/10.1016/j.clinthera.2014.07.017

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Arslan MN, Ozgun A, Das T, Kumru D, Sam B, Koc S (2016) Colchicine-induced rhabdomyolysis: an autopsy case. Am J Forensic Med Pathol 37:57–59. https://doi.org/10.1097/paf.0000000000000225

    Article  PubMed  Google Scholar 

  12. 12.

    Lee YM, Kaplan MM (2003) Efficacy of colchicine in patients with primary biliary cirrhosis poorly responsive to ursodiol and methotrexate. Am J Gastroenterol 98:205–208. https://doi.org/10.1111/j.1572-0241.2003.07182.x

    Article  PubMed  Google Scholar 

  13. 13.

    Slobodnick A, Shah B, Pillinger MH, Krasnokutsky S (2015) Colchicine: old and new. Am J Med 128:461–470. https://doi.org/10.1016/j.amjmed.2014.12.010

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Sannohe S, Makino Y, Kita T, Kuroda N, Shinozuka T (2002) Colchicine poisoning resulting from accidental ingestion of meadow saffron (Colchicum autumnale). J Forensic Sci 47:1391–1396

    CAS  Article  Google Scholar 

  15. 15.

    Sundov Z, Nincevic Z, Definis-Gojanovic M, Glavina-Durdov M, Jukic I, Hulina N, Tonkic A (2005) Fatal colchicine poisoning by accidental ingestion of meadow saffron-case report. Forensic Sci Int 149:253–256. https://doi.org/10.1016/j.forsciint.2004.06.034

    Article  PubMed  Google Scholar 

  16. 16.

    Wollersen H, Erdmann F, Risse M, Dettmeyer R (2009) Accidental fatal ingestion of colchicine-containing leaves--toxicological and histological findings. Leg Med (Tokyo) 11 Suppl 1:S498–S499. https://doi.org/10.1016/j.legalmed.2009.01.080

    Article  Google Scholar 

  17. 17.

    Babu YP, Manjunath S, Bakkannavar SM, Acharya J, Kumar GN (2012) Colchicine poisoning: a case report and review of literature. Med Sci Law 52:205–206. https://doi.org/10.1258/msl.2012.012025

    Article  PubMed  Google Scholar 

  18. 18.

    Deveaux M, Hubert N, Demarly C (2004) Colchicine poisoning: case report of two suicides. Forensic Sci Int 143:219–222. https://doi.org/10.1016/j.forsciint.2004.02.040

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Kande Vidanalage CJ, Ekanayeka R, Wijewardane DK (2016) Case report: a rare case of attempted homicide with Gloriosa superba seeds. BMC Pharmacol Toxicol 17:26. https://doi.org/10.1186/s40360-016-0069-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Plant toxicological information: the Japanese Ministry of Health, Labor and Welfare. https://www.mhlw.go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/shokuhin/yuudoku/. Accessed 4 Nov 2018

  21. 21.

    Klintschar M, Beham-Schmidt C, Radner H, Henning G, Roll P (1999) Colchicine poisoning by accidental ingestion of meadow saffron (Colchicum autumnale): pathological and medicolegal aspects. Forensic Sci Int 106:191–200

    CAS  Article  Google Scholar 

  22. 22.

    Byard RW, Stockham PC, Gilbert JD (2005) Vomiting, diarrhea, and sudden death with recent southeast Asian travel: fatal colchicine toxicity. Forensic Sci Med Pathol 1:149–152. https://doi.org/10.1385/fsmp:1:2:149

    Article  PubMed  Google Scholar 

  23. 23.

    Lauer E, Widmer C, Versace F, Staub C, Mangin P, Sabatasso S, Augsburger M, Déglon J (2013) Body fluid and tissue analysis using filter paper sampling support prior to LC-MS/MS: application to fatal overdose with colchicine. Drug Test Anal 5:763–772. https://doi.org/10.1002/dta.1496

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Aghabiklooei A, Zamani N, Hassanian-Moghaddam H, Nasouhi S, Mashayekhian M (2014) Acute colchicine overdose: report of three cases. Reumatismo 65:307–311. https://doi.org/10.4081/reumatismo.2013.720

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Sucher NJ, Hennell JR, Carles MC (2012) DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants. Methods Mol Biol 862:13–22. https://doi.org/10.1007/978-1-61779-609-8_2

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Houston R, Birck M, Hughes-Stamm S, Gangitano D (2016) Evaluation of a 13-loci STR multiplex system for Cannabis sativa genetic identification. Int J Legal Med 130:635–647. https://doi.org/10.1007/s00414-015-1296-x

    Article  PubMed  Google Scholar 

  27. 27.

    Matsuyama S, Nishi K (2011) Genus identification of toxic plant by real-time PCR. Int J Legal Med 125:211–217. https://doi.org/10.1007/s00414-010-0487-8

    Article  PubMed  Google Scholar 

  28. 28.

    Nguyen PA, Kim JS, Kim JH (2015) The complete chloroplast genome of colchicine plants (Colchicum autumnale L. and Gloriosa superba L.) and its application for identifying the genus. Planta 242:223–237. https://doi.org/10.1007/s00425-015-2303-7

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Ishida N, Sakurada M, Kusunoki H, Ueno Y (2018) Development of a simultaneous identification method for 13 animal species using two multiplex real-time PCR assays and melting curve analysis. Leg Med (Tokyo) 30:64–71. https://doi.org/10.1016/j.legalmed.2017.11.007

    CAS  Article  Google Scholar 

  30. 30.

    Tsuruda S, Akaki K, Hiwaki H, Suzuki A, Akiyama H (2012) Multiplex real-time PCR assay for simultaneous detection of Omphalotus guepiniformis and Lentinula edodes. Biosci Biotechnol Biochem 76:1343–1349. https://doi.org/10.1271/bbb.120090

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Grube S, Schonling J, Prange A (2015) Evaluation of a triplex real-time PCR system to detect the plant-pathogenic molds Alternaria spp., Fusarium spp. and C. purpurea. J Microbiol Methods 119:180–188. https://doi.org/10.1016/j.mimet.2015.10.017

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Yamanoi E, Uchiyama S, Sakurada M, Ueno Y (2018) sjTREC quantification using SYBR quantitative PCR for age estimation of bloodstains in a Japanese population. Leg Med (Tokyo) 32:71–74. https://doi.org/10.1016/j.legalmed.2018.03.003

    CAS  Article  Google Scholar 

  33. 33.

    Walid Turk SO (2015) Determination of colchicine contents in different Jordanian colchicum spp. Jordan J Pharm Sci 8:119–122

    Article  Google Scholar 

  34. 34.

    Maroyi A (2012) Phylogenetic relationships in genus Gloriosa L. Afr J Biotechnol 11:14732–14737. https://doi.org/10.5897/AJB12.1522

    Article  Google Scholar 

  35. 35.

    Okuda K, Maseda C, Asari M, Yamada H, Ohtani S, Ogawa K, Adachi Y, Kiya H, Yokota J, Saito M, Shimizu K (2015) An autopsy case of colchicine poisoning by Colchicine autumnale. Res Pract Forens Med 58:13–20

    Google Scholar 

  36. 36.

    Ferner RE, Aronson JK (2018) The toxicological significance of post-mortem drug concentrations in bile. Clin Toxicol (Phila) 56:7–14. https://doi.org/10.1080/15563650.2017.1339886

    CAS  Article  Google Scholar 

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Acknowledgments

We thank Edanz Group (www.edanzediting.com/ac) for editing the English text of a draft of this manuscript.

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Correspondence to Makoto Sakurada.

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Informed consent was obtained, and autopsy sample collection was conducted with the permission of the Ethics Committee of the Kobe University Graduate School of Medicine (#1799).

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Sakurada, M., Yoshioka, N., Kuse, A. et al. Rapid identification of Gloriosa superba and Colchicum autumnale by melting curve analysis: application to a suicide case involving massive ingestion of G. superba. Int J Legal Med 133, 1065–1073 (2019). https://doi.org/10.1007/s00414-019-02060-x

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Keywords

  • Real-time PCR
  • Melting curve analysis
  • Colchicine
  • Gloriosa superba
  • Colchicum autumnale
  • Forensic analysis of stomach contents