Presentation number: FG 8
ANALYSIS OF CANNABINOID PROFILES AND THCAS SEQUENCES IN SEIZED CANNABIS RECOVERED FROM GROWING FARM
Lucija Barbarić, Slavena Cukrov Bezbradica, Ines Gmajnički, Adela Makar, Andrea Ledić
Forensic Science Centre “Ivan Vučetić”, Zagreb, Croatia
The aim of this study was to assess the genetic and chemical profiles of the seized cannabis obtained from the growing farm. According to the farmer, certified variety Santhica 27 was sown. However, police officers recovered 118 plants under the suspicion of drug production. Quantitative cannabinoid content was determined using GC-FID on Agilent 7890A GC System. 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 using Qiagen Multiplex PCR kit. Sequencing was performed according to the laboratory standard operating procedures using Big Dye Terminator v.3.1 Cycle Sequencing Kit on AB3500xl GA (both from Life Technologies). Data analysis was accomplished in Sequencher v.5.4.6 (Gene Codes). Sequences were aligned against the THCAS coding sequence of the drug-type Skunk deposited in NCBI (KJ469378). Based on qualitative chemotype, 61% of total samples were cannabigerol (CBG) predominant, and 39% were THC dominant. Alignment of the obtained sequences correlated with their cannabinoid content, clustering in two distinct groups. Eleven identical sequences differed from variants in hemp Finola and probably Santhica 27 that showed undetectable THCAS. Moreover, several variants were revealed including 1064A that had been previously reported to be responsible for THCAS inactivation and cannabigerolic acid (CBGA) precursor accumulation. This finding was in correlation with the average CBG content of 2%. The remaining three sequences were identical to those obtained in drug-type. The average proportion of THC in these plants was 2.5%. According to chemical and genetic results, we conclude that on the industrial hemp farm was growing THC and CBG predominant cannabis varieties under the guise of certified variety Santhica 27.
Key words: Cannabis sativa L, chemotype, THCA synthase, growing farm
Presentation number: FG 9
DNA ANALYSIS OF SCARCE FUNGAL SAMPLES INDICATES THEIR PSYCHOACTIVE ORIGIN
Ivana Horjan Zanki, Andrea Ledić, Adela Makar
Forensic Science Centre “Ivan Vučetić”, Ministry of the Interior, Zagreb, Croatia
The majority of psychoactive fungi contain psilocybin (PY) and psilocin (PI), latter causes LSD-like psychoactive effects but lesser in intensity. PY and PI are illegal in Croatia and most of EU countries, while fungi as their source are mainly accepted. Since PY/PI are not yet synthesized in fungal spores and sclerotia (“truffles”), drug users increasingly buy them via Internet without legal consequences. Furthermore, established toxicology and morphology methods are useless on these kind of samples. Hence, main goal of this study was to present the usefulness of DNA analysis on abovementioned forensic samples in order to confirm its psychoactive origin. Study addressed sequencing of internal transcribed spacer (ITS) of nuclear DNA and large portion of 28S gene (nuclear-encoded large subunit ribosomal RNA genes, nLSU-rDNA). ITS region enables unambiguous identification of fungal species while part of LSU gene from 5′ end enables distinguishing between closely related psychoactive and non-psychoactive species. DNA from total of 14 “spore-print“and „truffle“ samples seized on Croatian territory (2010-2014), were isolated and ITS region and large portion of 28S gene (nLSU-rDNA) were sequenced. ITS and LSU sequences were obtained from 11 out of 14 tested samples with haplotypes congruent to GenBank database sequences of psychoactive species Psilocybe cubensis and Psilocybe mexicana. One out of 14 samples most probably represents a mixture of these two, and for the two remaining samples only ITS haplotype was obtained, matching those of P. cubensis. As previously published, P. cubensis varieties are most popular among users due to their proven potency and are easy growing. Applied DNA method can be easily implemented into routine forensic workflow. The latter legislation update, i.e. to include fungi species/body parts as the source of psychoactive substances could lead to continuing decrease of illegal fungi drug abuse.
Key words: „spore print“, „truffles“, DNA sequencing, ITS, LSU
Presentation number: FG 10
IDENTIFICATION OF GENETIC MATERIAL OF WATERBORNE MICROORGANISMS FROM STERNAL ASPIRATE
Karola Petrus, Vivien Fejes, Dominika Szűcs, Katalin Sipos, Gábor Simon, Viktor Soma Poór
University of Pécs, Medical School, Department of Forensic Medicin, Pécs, Hungary
The most widely used complementary method for the diagnosis of drowning is the detection of waterborne organisms in the organs of systemic circulation. In this study, we adopted sternal bone marrow aspiration – which in hematology a routine method – instead of femoral bone marrow opening, to prevent the possible contamination during autopsy. During autopsy, besides sternum aspirate, lung and spleen tissues, and bone marrow of the femur were also taken for test the presence of diatom shells by microscopy, and cyanobacterial DNA by PCR. From all tissue samples, 2 g were mixed with 6 ml digesting buffer (0.01 M Tris, 2 % SDS) and 20 µl Proteinase K. After 2 days of incubation at 56°C, microscopic slides were prepared for diatom test, furthermore, 400 µl aliquots were saved for cyanobacterial DNA detection. DNA extraction was carried out with Macherey-Nagel NucleoSpin Soil kit, followed by amplification of a segment of cyanobacterial 16S rRNA gene. The amplicons were separated in 2 % agarose gel and visualized with SYBR Gold intercalating dye. We were able to obtain sternal bone marrow aspirate in all the tested seven suspected drowning cases. In four of the sternal samples, both diatoms and cyanobacterial DNA were detected, while in one additional case, sternum was tested positive by PCR, but no diatom shells were identified. Femoral bone marrow was positive for diatoms only in one case, and by PCR in two cases. The rest of the cases – drowning into a bathtub, falling into a cistern -, all tissue samples were negative. According to autopsy, heart failure, and the high fall was the cause of death, respectively. Sternal bone marrow aspiration can be simply performed in the beginning of the autopsy, before the body cavity is opened, therefore minimizes the chance of contamination. Our results showed that diatom test has low sensitivity, which can be increased with PCR-based methods. Sternal bone marrow was found to be a better source for detection of waterborne organisms in our pilot study than the femoral bone marrow.
Key words: Drowning, Cyanobacteria, DNA