Technical noteIdentification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe
Introduction
Cannabis sativa L. belongs to the Cannabaceae family, and is one of the most widely used and long known illicit drugs in the world. The Δ9-tetrahydrocannabinol (Δ9-THC) is the major cannabinoid unique to C. sativa that acts as the principle psychoactive compound [1]. The key enzyme involved in the synthesis of tetrahydrocannabinolic acid (THCA), the THC parent compound, is the oxidoreductase THCA synthase, which converts cannabigerolic acid (CBGA) into THCA. THCA could then be converted to THC upon heating or smoking [2].
Although identification of C. sativa can be achieved by chemical and botanical examinations [3], each of these methods has different requirements and limitations. A considerable amount of questioned plant materials is required for sample preparation in qualitative and quantitative chromatographic separation analyses of Δ9-THC [4]. Moreover, the level of Δ9-THC also depends upon the plant growth stage; immature plants have no or very low THC level and would reach the highest level at the flowering stage [5]. Botanical identification is based on the examination of plant's macro- and micro-morphology, which requires expertise of botanist. Positive identification cannot be confirmed if the questioned material contained insufficient botanical characteristics. Alternatively, C. sativa can be identified by PCR-based methodology using molecular DNA markers, such as the nuclear ITS, THCAS, STR markers, and the chloroplast trnL-F marker [6], [7], [8], [9]. Quality and quantity of the DNA extract directly affect the success of PCR analysis. DNA extraction is a critical prerequisite step prior to PCR analysis. Recovering DNA from limited and/or trace amount of questioned plant materials often becomes an obstacle for PCR analysis. Although plant direct PCR kit has been developed to save time, reduce DNA loss, and reduce risk of cross contamination, the method is not robust for handling various plant species with different natures [10].
In situ hybridization [11] is a cyto-molecular genetics technique that utilizes target-specific, easy to penetrate cell and/or tissue nucleotide probe to detect its complementary target DNA region by hybridization. By attaching fluorescent reporter molecule to the probe, sensitive detection can be enhanced, and hence the term fluorescence in situ hybridization (FISH). This allows visualization of the successful hybridization through fluorescence microscopy. Nucleotide probes could be prepared by either direct or indirect labeling methods [12]. To conduct in situ hybridization experiments, cell or tissue would be fixed on a microscope slide. Combining the denatured probe and target would then allow annealing of complementary nucleotide sequences, or hybridization. If indirectly labeled probe is used, there would be extra steps in the post-hybridization process for enzymatic or immunological detection system. Though the method is lengthier than that of directly labeled probes, the use of indirectly labeled probe has the advantage of signal amplification by incorporating higher number of fluorescent reporter molecule through PCR, or by using several layers of antibodies, providing stronger FISH signals. The technique could be applied to chromosomes in both interphase and metaphase stages [13].
FISH has been widely applied to detect and identify species, especially microorganisms [14]. This technique has also been well utilized in forensic examinations of sexual assaults and sex-related evidence to detect the presence of male cells in male/female mixtures [15], [16], [17]. It has also been applied to sex-type telogen head hair [18]. Previous work also showed that FISH can be applied to specimens ranging from fossils to fresh specimens [12]. However, there was no reported cyto-molecular genetics method for forensic detection of C. sativa to date.
In cases of C. sativa seizure or crime scene investigation involving suspected narcotic plant materials, the evidence in question may be present in the form of dried, mixed powder, and could be available in very small amount, such as trace botanical materials on wrap. These difficulties limit the use of valid analysis/examination to confirm the presence of C. sativa L. [4], [19]. FISH would be a suitable technique that can be developed to analyze such difficult samples, as, unlike PCR, it does not require DNA extraction from its source. Furthermore, the use of target-specific probe allows for highly specific detection, and the target cells identified on the microscope slide could be isolated by micro-dissection for further analysis.
This study aimed to develop FISH probe for the identification of C. sativa L. It is reported here that the newly designed 1-kb THCA synthase hybridization probe demonstrated to be Cannabis-specific and could be applied to the analysis of both fresh and dried C. sativa materials.
Section snippets
Plant samples
Leaves were collected from 22 individual Cannabis sativa L. plants, which were grown from seeds in vitro and soil-potted. For dried samples, leaves were dried in an oven at 42 °C and ground to fine powder. Leaves were also collected from the following non-target plants for probe specificity testing: Humulus lupulus (common hop), a closely-related member in the Cannabaceae family; Papaver sp. (member of poppy family) and Mitragyna speciosa (Kratom), both are classified as narcotic plant in Thai
Specificity and reproducibility of the 1-kb THCA synthase and 700-bp cannabis-ITS probe detection
When the 1-kb THCA synthase and 700-bp Cannabis-ITS biotin-labeled probes were tested against C. sativa, H. lupulus, Papaver sp., N. tabacum, and M. speciosa genomic DNA samples, the dot-blot analysis results showed no hybridization signal. This may be due to the intactness of genomic DNA that hindered probes' access to the target DNA region. Therefore, the target region was then enriched by PCR using the Can_05/Can_B and universal ITS4/ITS5 primer pairs. Analysis of PCR products by ethidium
Discussion
The 1-kb THCAS hybridization probe was designed from the 1.6-kb cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene sequence, which is unique to the C. sativa species. As a result, no hybridization signal was obtained when other plant species were analyzed in the blot analysis and FISH experiments. These experimental results confirmed the Cannabis-specificity and reproducibility of detection by the 1-kb THCA synthase hybridization probe.
As the THCAS gene is present in all C.
Conclusions
This study provided an alternative tool for the identification of Cannabis cells through the use of FISH and the 1-kb THCA synthase hybridization probe. The results obtained showed that the technique was Cannabis-specific, repeatable, and reproducible. Detection of the presence/absence of FISH signals using the 1-kb THCAS Cannabis-specific hybridization probe would help to quickly identify Cannabis cells, especially when trace amount of evidential material is available and no other test could
Conflicts of interest
None.
Acknowledgement
Pattraporn Jeangkhwoa was sponsored by the Development and Promotion of Science and Technology Talent Project (DPST). Authors would also like to acknowledge the Olympus Bioimaging Center (OBC), Faculty of Science, Mahidol University for the imaging facilities.
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