title: Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.
abstract: 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.
authors.affiliation: Laboratory of Molecular Pharmacognosy, Division of Pharmaceutical Sciences, Graduate School of Medical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, 920-1192, Japan.|Forensic Science Laboratory, Ishikawa Prefectural Police H.Q., 1-1 Kuratsuki, Kanazawa, Ishikawa, 920-8553, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kitamura
authors.affiliation: Medicinal Plant Garden, Tokyo Metropolitan Institute of Public Health, 21-1 Nakajima-cho, Kodaira-Shi, Tokyo, 187-0033, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Aragane
authors.affiliation: Medicinal Plant Garden, Tokyo Metropolitan Institute of Public Health, 21-1 Nakajima-cho, Kodaira-Shi, Tokyo, 187-0033, Japan.
authors.forename: Kou
authors.initials: K
authors.lastname: Nakamura
authors.affiliation: Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka, 815-8511, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Watanabe
authors.affiliation: Laboratory of Molecular Pharmacognosy, Division of Pharmaceutical Sciences, Graduate School of Medical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, 920-1192, Japan. sasaki@p.kanazawa-u.ac.jp.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Sasaki
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002188:Cannabis / Q000737:chemistry*
keywords: Cannabis sativa; Isothermal amplification; LAMP; Marijuana; Melting curve analysis; Tetrahydrocannabinolic acid synthase gene