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Cannabis sativa L. is the most commonly seized illicit drug in Croatia. Recent lifting of restrictions has led to a new problem for the law enforcement: prevention of illegal production and sale in a rapidly advancing cannabis industry that offers a wide range of commercial products. Croatian law allows production of cannabis plant varieties listed in the European Union Plant Variety Catalogue (EU PVC) with less than 0.2% w/w (weight/weight) of total Δ9-tetrahydrocannabinol (THC) content in the dry plant material. Hence, there is a particular need to study the commercial cannabis products. The aim of this study is to assess the genetic and chemical analyses of seizures obtained from CBD stores and to compare the results with the product declaration. Product declarations do not indicate the Cannabis variety, but state that the variety is listed in the EU PVC with the THC content less than 0.2%. Cannabis samples were obtained from 46 seizures confiscated in the last year. Quantitative cannabinoids analysis was performed by GC-FID on Agilent 7890A GC System according to the accredited workflow. Extraction of DNA from plant material was performed using DNAeasy Plant Mini kit (Qiagen) following the manufacturer’s protocol. DNA quantity was determined by Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific). A portion of the tetrahydrocannabinolic acid synthase (THCAS) gene was amplified with primers “g” and “h” as outlined in (1) with the following modification. Briefly, protocol was optimized with duplex primer set using Qiagen Multiplex PCR kit. Furthermore, samples with detected THCAS marker were subjected to the D589 marker analysis with primers described in (2). PCR products were separated by capillary electrophoresis on AB 3500 GA. THCAS gene portion sequencing (265-1490bp) was conducted for samples with both markers detected along with two drug- type (positive controls) and two certified fiber- type varieties (negative controls). Sequencing was performed according to the laboratory standard operating procedures with primers described in (1) using Big Dye Terminator v.3.1 Cycle Sequencing Kit on AB3500xl GA (both from Life Technologies) and data analysis in Sequencher v.5.4.6. Sequences were aligned against the THCAS coding sequence of the drug-type cultivar Skunk (GenBank ID KJ469378). Based on qualitative chemotype, 91.3% of samples were CBD (cannabidiol) predominant, and the remaining samples were CBG (cannabigerol) dominant. In 17 out of 46 tested samples, THC content was higher than 0.2 %. THCAS and D589 markers were detected in 12 analyzed samples. Eleven identical THCAS sequences were determined, which differed from the variants obtained in fiber-type except at the position 749. Moreover, G to A SNP at the 1064bp was observed. Moreover, one sample showed the sequence identical to the drug- type. In 37% of total samples, THC content was above the value specified in product declaration. THCAS and D589 markers (specific for drug type varieties) were determined in 26.1% of tested samples. The complete concordance between abovementioned markers was observed, however their detection was not in agreement with the qualitative chemotype. Subsequent analysis demonstrated differences in THCAS sequences in comparison with the hemp sequences. Interestingly, 1064G→A substitution has been reported as a marker for THCA inactivation and CBGA dominance (3). However, this finding was not in correlation with the CBG content since it was found in only four tested samples. Hence, future studies will be focused on genetic analysis of complete THCAS and CBDAS genes in certified varieties and seizures. |
