Journal of Natural Medicines

, Volume 71, Issue 1, pp 86–95 | Cite as

Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay

  • Masashi Kitamura
  • Masako Aragane
  • Kou Nakamura
  • Kazuhito Watanabe
  • Yohei Sasaki
Original Paper


In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content—THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.


Cannabis sativa Isothermal amplification Tetrahydrocannabinolic acid synthase gene Melting curve analysis LAMP Marijuana 



We thank Kaneka Corporation for technical advice and our colleagues for support extended in the course of this study.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. 1.
    House JD, Neufeld J, Leson G (2010) Evaluating the quality of protein from hemp seed (Cannabis sativa L.) products through the use of the protein digestibility-corrected amino acid score method. J Agric Food Chem 58:11801–11807CrossRefPubMedGoogle Scholar
  2. 2.
    Callaway J, Schwab U, Harvima I, Halonen P, Mykkänen O, Hyvönen P, Järvinen T (2005) Efficacy of dietary hempseed oil in patients with atopic dermatitis. J Dermatolog Treat 16:87–94CrossRefPubMedGoogle Scholar
  3. 3.
    Simopoulos AP (2008) The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. Exp Biol Med (Maywood) 233:674–688CrossRefGoogle Scholar
  4. 4.
    Kreuger E, Sipos B, Zacchi G, Svensson SE, Björnsson L (2011) Bioconversion of industrial hemp to ethanol and methane: the benefits of steam pretreatment and co-production. Bioresour Technol 102:3457–3465CrossRefPubMedGoogle Scholar
  5. 5.
    Kuglarz M, Alvarado-Morales M, Karakashev D, Angelidaki I (2016) Integrated production of cellulosic bioethanol and succinic acid from industrial hemp in a biorefinery concept. Bioresour Technol 200:639–647CrossRefPubMedGoogle Scholar
  6. 6.
    Press CA, Knupp KG, Chapman KE (2015) Parental reporting of response to oral cannabis extracts for treatment of refractory epilepsy. Epilepsy Behav 45:49–52CrossRefPubMedGoogle Scholar
  7. 7.
    Kevorkian S, Bonn-Miller MO, Belendiuk K, Carney DM, Roberson-Nay R, Berenz EC (2015) Associations among trauma, posttraumatic stress disorder, cannabis use, and cannabis use disorder in a nationally representative epidemiologic sample. Psychol Addict Behav 29:633–638CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Abrams DI, Jay CA, Shade SB, Vizoso H, Reda H, Press S, Kelly ME, Rowbotham MC, Petersen KL (2007) Cannabis in painful HIV-associated sensory neuropathy: a randomized placebo-controlled trial. Neurology 68:515–521CrossRefPubMedGoogle Scholar
  9. 9.
    Naftali T, Lev LB, Yablecovitch D, Yablekovitz D, Half E, Konikoff FM (2011) Treatment of Crohn’s disease with cannabis: an observational study. Isr Med Assoc J 13:455–458PubMedGoogle Scholar
  10. 10.
    Ahmed SA, Ross SA, Slade D, Radwan MM, Zulfiqar F, Matsumoto RR, Xu YT, Viard E, Speth RC, Karamyan VT, ElSohly MA (2008) Cannabinoid ester constituents from high-potency Cannabis sativa. J Nat Prod 71:536–542CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Grotenhermen F (2003) Pharmacokinetics and pharmacodynamics of cannabinoids. Clin Pharmacokinet 42:327–360CrossRefPubMedGoogle Scholar
  12. 12.
    de Meijer EP, Bagatta M, Carboni A, Crucitti P, Moliterni VM, Ranalli P, Mandolino G (2003) The inheritance of chemical phenotype in Cannabis sativa L. Genetics 163:335–346PubMedPubMedCentralGoogle Scholar
  13. 13.
    Kojoma M, Seki H, Yoshida S, Muranaka T (2006) DNA polymorphisms in the tetrahydrocannabinolic acid (THCA) synthase gene in drug-type and fiber-type Cannabis sativa L. Forensic Sci Int 159:132–140CrossRefPubMedGoogle Scholar
  14. 14.
    Hillig KW, Mahlberg PG (2004) A chemotaxonomic analysis of cannabinoid variation in Cannabis (Cannabaceae). Am J Bot 91:966–975CrossRefPubMedGoogle Scholar
  15. 15.
    Omar J, Olivares M, Alzaga M, Etxebarria N (2013) Optimisation and characterisation of marihuana extracts obtained by supercritical fluid extraction and focused ultrasound extraction and retention time locking GC–MS. J Sep Sci 36:1397–1404CrossRefPubMedGoogle Scholar
  16. 16.
    Aizpurua-Olaizola O, Omar J, Navarro P, Olivares M, Etxebarria N, Usobiaga A (2014) Identification and quantification of cannabinoids in Cannabis sativa L. plants by high performance liquid chromatography–mass spectrometry. Anal Bioanal Chem 406:7549–7560CrossRefPubMedGoogle Scholar
  17. 17.
    Grauwiler SB, Scholer A, Drewe J (2007) Development of a LC/MS/MS method for the analysis of cannabinoids in human EDTA-plasma and urine after small doses of Cannabis sativa extracts. J Chromatogr B Anal Technol Biomed Life Sci 850:515–522CrossRefGoogle Scholar
  18. 18.
    Vanhove W, Van Damme P, Meert N (2011) Factors determining yield and quality of illicit indoor cannabis (Cannabis spp.) production. Forensic Sci Int 212:158–163CrossRefPubMedGoogle Scholar
  19. 19.
    Kim E, Mahlberg P (1997) Immunochemical localization of tetrahydrocannabinol (THC) in cryofixed glandular trichomes of Cannabis (Cannabaceae). Am J Bot 84:336CrossRefPubMedGoogle Scholar
  20. 20.
    Ross SA, Mehmedic Z, Murphy TP, Elsohly MA (2000) GC–MS analysis of the total δ9-THC content of both drug- and fiber-type cannabis seeds. J Anal Toxicol 24:715–717CrossRefPubMedGoogle Scholar
  21. 21.
    Miller Coyle H, Palmbach T, Juliano N, Ladd C, Lee HC (2003) An overview of DNA methods for the identification and individualization of marijuana. Croat Med J 44:315–321PubMedGoogle Scholar
  22. 22.
    Toonen M, Ribot S, Thissen J (2006) Yield of illicit indoor cannabis cultivation in the Netherlands. J Forensic Sci 51:1050–1054CrossRefPubMedGoogle Scholar
  23. 23.
    Sirikantaramas S, Taura F, Tanaka Y, Ishikawa Y, Morimoto S, Shoyama Y (2005) Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is secreted into the storage cavity of the glandular trichomes. Plant Cell Physiol 46:1578–1582CrossRefPubMedGoogle Scholar
  24. 24.
    Taura F, Dono E, Sirikantaramas S, Yoshimura K, Shoyama Y, Morimoto S (2007) Production of Δ1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris. Biochem Biophys Res Commun 361:675–680CrossRefPubMedGoogle Scholar
  25. 25.
    Sirikantaramas S, Morimoto S, Shoyama Y, Ishikawa Y, Wada Y, Taura F (2004) The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Δ1-tetrahydrocannabinolic acid synthase from Cannabis sativa L. J Biol Chem 279:39767–39774CrossRefPubMedGoogle Scholar
  26. 26.
    Rotherham D, Harbison SA (2011) Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay. Forensic Sci Int 207:193–197CrossRefPubMedGoogle Scholar
  27. 27.
    Staginnus C, Zörntlein S, de Meijer E (2014) A PCR marker linked to a THCA synthase polymorphism is a reliable tool to discriminate potentially THC-rich plants of Cannabis sativa L. J Forensic Sci 59:919–926CrossRefPubMedGoogle Scholar
  28. 28.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Mori Y, Nagamine K, Tomita N, Notomi T (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289:150–154CrossRefPubMedGoogle Scholar
  30. 30.
    Kurosaki Y, Takada A, Ebihara H, Grolla A, Kamo N, Feldmann H, Kawaoka Y, Yasuda J (2007) Rapid and simple detection of Ebola virus by reverse transcription-loop-mediated isothermal amplification. J Virol Methods 141:78–83CrossRefPubMedGoogle Scholar
  31. 31.
    Ravan H, Amandadi M, Sanadgol N (2016) A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157: H7. Microb Pathog 91:161–165CrossRefPubMedGoogle Scholar
  32. 32.
    Sasaki Y, Nagumo S (2007) Rapid identification of Curcuma longa and C. aromatica by LAMP. Biol Pharm Bull 30:2229–2230CrossRefPubMedGoogle Scholar
  33. 33.
    Sasaki Y, Komatsu K, Nagumo S (2008) Rapid detection of Panax ginseng by loop-mediated isothermal amplification and its application to authentication of Ginseng. Biol Pharm Bull 31:1806–1808CrossRefPubMedGoogle Scholar
  34. 34.
    Sasaki Y, Fujimoto T, Aragane M, Yasuda I, Nagumo S (2009) Rapid and sensitive detection of Lophophora williamsii by loop-mediated isothermal amplification. Biol Pharm Bull 32:887–891CrossRefPubMedGoogle Scholar
  35. 35.
    Kitamura M, Aragane M, Nakamura K, Watanabe K, Sasaki Y (2016) Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa. Biol Pharm Bull 39:1144–1149CrossRefPubMedGoogle Scholar
  36. 36.
    Shoyama Y, Tamada T, Kurihara K, Takeuchi A, Taura F, Arai S, Blaber M, Morimoto S, Kuroki R (2012) Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa. J Mol Biol 423:96–105CrossRefPubMedGoogle Scholar
  37. 37.
    Alaoui MA, Ibrahimi A, Semlali O, Tarhda Z, Marouane M, Najwa A, Soulaymani A, Fahime EE (2014) Affinity comparison of different THCA synthase to CBGA using modeling computational approaches. Bioinformation 10:33–38CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Sawler J, Stout JM, Gardner KM, Hudson D, Vidmar J, Butler L, Page JE, Myles S (2015) The genetic structure of marijuana and hemp. PLoS One 10:e0133292CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    van Bakel H, Stout JM, Cote AG, Tallon CM, Sharpe AG, Hughes TR, Page JE (2011) The draft genome and transcriptome of Cannabis sativa. Genome Biol 12:R102CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Monden Y, Takasaki K, Futo S, Niwa K, Kawase M, Akitake H, Tahara M (2014) A rapid and enhanced DNA detection method for crop cultivar discrimination. J Biotechnol 185:57–62CrossRefPubMedGoogle Scholar
  41. 41.
    Kaneko H, Kawana T, Fukushima E, Suzutani T (2007) Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 70:499–501CrossRefPubMedGoogle Scholar

Copyright information

© The Japanese Society of Pharmacognosy and Springer Japan 2016

Authors and Affiliations

  • Masashi Kitamura
    • 1
    • 2
  • Masako Aragane
    • 3
  • Kou Nakamura
    • 3
  • Kazuhito Watanabe
    • 4
  • Yohei Sasaki
    • 1
  1. 1.Laboratory of Molecular Pharmacognosy, Division of Pharmaceutical Sciences, Graduate School of Medical SciencesKanazawa UniversityKanazawaJapan
  2. 2.Forensic Science LaboratoryIshikawa Prefectural Police H.Q.KanazawaJapan
  3. 3.Medicinal Plant GardenTokyo Metropolitan Institute of Public HealthKodaira-ShiJapan
  4. 4.Daiichi University of PharmacyFukuokaJapan

Personalised recommendations