title: Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.
abstract: This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace.
authors.affiliation: M.Sc. Program in Plant Science, Department of Plant Science, Faculty of Science and Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University, Thailand.
authors.forename: Pattraporn
authors.initials: P
authors.lastname: Jeangkhwoa
authors.affiliation: Forensic Science Program, Faculty of Science, Mahidol University, Thailand.
authors.forename: Achirapa
authors.initials: A
authors.lastname: Bandhaya
authors.affiliation: Department of Plant Science, Faculty of Science, Mahidol University, Thailand.
authors.forename: Puangpaka
authors.initials: P
authors.lastname: Umpunjun
authors.affiliation: Department of Plant Science, Faculty of Science, Mahidol University, Thailand.
authors.forename: Ngarmnij
authors.initials: N
authors.lastname: Chuenboonngarm
authors.affiliation: Forensic Science Program, Faculty of Science, Mahidol University, Thailand; Department of Plant Science, Faculty of Science, Mahidol University, Thailand. Electronic address: nathinee.pan@mahidol.ac.th.
authors.forename: Nathinee
authors.initials: N
authors.lastname: Panvisavas
mesh_terms: D002188:Cannabis / Q000235:genetics*; D053593:Forensic Toxicology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D019746:Intramolecular Oxidoreductases / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics*
chemical_list: D012335:RNA, Ribosomal; C088179:RNA, ribosomal, 45S; D019746:Intramolecular Oxidoreductases; C100678:cannabidiolic acid synthase
keywords: Cannabis sativa; Fluorescence in situ hybridization (FISH); THCA synthase gene