Patent Application
20210325411
The content of the electronically submitted sequence listing in ASCII text file 2020_33_US_SEQUENCE_LISTING.txt, created Oct. 23, 2020, with a size of 6,152 bytes is hereby incorporated by reference in its entirety.
The invention refers to the identification of plant samples. The present method comprises the molecular analysis of DNA markers and the determination of the chemical profile by means of 13C and 1H NMR resonance patterns, of cannabis samples to achieve their forensic identification and traceability. Short tandem repeat analysis (STR) provides knowledge about the genetics of the sample, and nuclear magnetic resonance (NMR) analyses the phenotypic characteristics of the sample.
Despite the long historical relationship between cannabis and human society, there is no in-depth knowledge of the chemo-taxonomy and chemical composition of this plant. Seventy unique compounds, known collectively as phytocannabinoids, have been identified in cannabis plants1.
There are estimated to be 104 naturally occurring cannabinoids in cannabis, the most studied ones being delta-9-tetrahydrocannabinol (Δ9-THC), delta-8-tetrahydrocannabinol THC), cannabidiol (CBD) and cannabinol (CBN)2.
Cannabis is commonly found in the following presentations:
There is currently a debate about the number of species included in the genus Cannabis. The genus is generally considered by the scientific community to consist of the species Cannabis sativa L and its subspecies: Cannabis sativa L sativa subspecies and Cannabis sativa L indica subspecies. Four varieties: Cannabis sativa L. sativa subspecies sativa variety; Cannabis sativa L. sativa subspecies spontaneous variety Vavilov; Cannabis sativa L. indica subspecies indica (Lam.) Wehmer variety; Cannabis sativa L. indica subspecies kafiristanica variety (Vavilov). Although the drug is considered to be monospecific (Cannabis sativa L)3, psychoactive substances can be obtained from all the above-mentioned species, subspecies and varieties.
In 2009, the Latin American Commission on Drugs and Democracy published a report that questioned the effectiveness of “prohibitionist policies based on the repression of production and interdiction of trafficking and distribution, as well as the criminalization of consumption, have not produced the expected results. “We are further away than ever from the proclaimed objective of eradicating drugs”4.
The country that pioneered the full legalization of the production and sale of cannabis for recreational use was Uruguay, which was later joined by numerous countries such as Canada, Jamaica, Australia, Georgia, the Netherlands and South Africa, all of which have different nuances in their regulation.
The acceptance of “medical cannabis” is more widespread. Its use is regulated in more than 40 countries, among which the following ones stand out: Germany, Brazil, Colombia, Croatia, Denmark, Estonia and United Kingdom among others.
Despite all these opening policies, none of these countries has managed to implement an effective method to completely eliminate or reduce to a minimum the entire parallel market to the legal/regulated one. For this reason, it is necessary to develop new tools for the different actors involved in the legalization/regulation of cannabis, either from a “recreational” or “medical” perspective.
One of these tools can be the creation of methods to correctly identify the source of a given sample of Cannabis. This could establish whether a given sample is from illegal origin, or from a variety that is legally traded.
Current methods can differentiate cannabis samples from other substances, but they are not specific enough to distinguish samples of cannabis from each other and to conclude whether another sample found later comes from the first variety tested, i.e. they are not useful for sample traceability.
DNA-only identification analyses are complicated in the case of plants, because for a sample from the same place of origin, there may be cross-breeding, pollination, climate, etc. and therefore, the genetic profile may be somewhat different from what is expected or known from the database5,8.
Furthermore, the chemical composition of a plant is derived from its specific environment. The chemical methods used to identify the samples and the origin of a cannabis sample include palynology, the study of the composition in spores and pollen7, the obtaining of the sample chemical profile8, or isotope analysis and the ratio of isotopes in samples, which are analyzed by mass spectrometry9,10.
On the other hand, the genetic methods found in the literature are based on Cannabis DNA amplification11,12,13,14, which has allowed forensic investigators to move away from the identification mentioned in the previous paragraph. These documents describe the use of short tandem repeats (STRs), single sequence repeats (SSRs) or microsatellites for the determination of a single DNA strand.
Document US2006/0035236 A1 discloses the use of STRs in multiplex DNA analysis for Cannabis samples determination15. However, sample prior separation in a polyacrylamide gel is required. All these analyses involve pre-treatment of the sample, which usually leads to decarboxylation of the terpenic derivatives and can result in loss of information and misidentification of a sample.
In the literature, information on the determination of the chemical composition of this plant has only been found by NMR techniques, either using two-dimensional NMR-1H techniques16 and the combined use of NMR-1H with HPLC, analyzed on the sample, after separation by chromatographic techniques17. However, in the present invention a 13C spectrum is also made, whose advantage is that there is a greater resolution between observed peaks, almost without signal overlapping. Signal overlapping is a common disadvantage in NMR-1H which leads to an increasing difficulty of the analysis. In addition, a greater range of frequencies can be analyzed, making the analysis more reliable.
What all the methods mentioned in the previous paragraphs have in common, regardless of the method used, is that their objective is the identification of cannabis samples with respect to other substances. However, the present invention is an improvement, as it allows the reliable and rapid identification of cannabis samples varieties with respect to other cannabis samples by means of fingerprint profiles.
The object of this invention is a rapid and standardized method that comprises molecular analysis techniques of DNA markers and the determination of the resonance pattern in NMR 13C and 1H, to obtain its fingerprint and thus determine its origin and traceability with respect to other cannabis samples. This allows the sample to be traced and distribution lines identified, achieving a degree of reliability in identification that exceeds that of any current analysis.
The method of the present invention has the following advantages:
The present invention refers to a method of obtaining a fingerprint of cannabis samples that comprises the following steps:
The term “sample” or “plant sample” in the present invention refers to any part of the cannabis plant, preferably leaves, stem, bulb, seeds or flowers, more preferably leaves. Such plant sample weighs between 0.1 and 10 g, preferably 0.2 and 1.0 g, more preferably 0.5 g.
The term “fingerprint” in the present invention refers to a combination of particular genetic and chemical characteristics which enable the identification and traceability of a sample of cannabis and differentiate it from other varieties of the same species.
Additionally, the method of the invention can differentiate plant samples from individuals of the same variety from other individuals of the same variety who have been subjected to particular and different environmental conditions.
The term “multiplex” is the ability to perform multiple simultaneous analyses in a single trial, the procedure for implementing such an ability in a process is called a “multiplexed trial”. It is applied in procedures that analyze several loci simultaneously.
The terms “markers” and “microsatellite markers” have equivalent meanings, referring to DNA sequences containing repetitive fragments, and are used interchangeably in the present invention.
Any other term used in the present specification shall have the usual meaning in the field of technology to which the present invention refers.
In the method of the invention the steps a) and b), and according to claim 1, do not have to be carried out in the order indicated. That is to say, it is not a chronological order, they can be carried out in the mentioned order or vice versa.
The STR analyzed in the method of the present invention are distributed throughout the genome and can preferably be selected among the following STR: D02, C11, H09, B01, E07, 305, 308, B05, H06, 501, CS1, 302, 301, B02, H11 and combinations thereof. Table 1 shows the GenBank database access numbers:
The step of determining the genetic profile of the cannabis sample from at least one STR comprises the following stages:
These steps can be performed by any means known in the state of the art, preferably by multiplexed PCR or individual marker PCR.
Preferably, the results of individual PCRs can be analyzed by agarose gels and the results of multiplexed PCR by capillary electrophoresis.
A further realization of the invention involves the pre-freezing at a temperature between −4° C. to −80° C. of the sample from which the STR are to be determined until its extraction.
A further embodiment of the invention comprises the detection of the presence of the Rubisco gene in cannabis samples prior to the determination of the genetic profile by STR marker analysis to confirm that the sample is a plant sample.
The presence of the Rubisco gene can be checked by sequencing and with any known state-of-the-art method involving amplification, Sanger sequencing and comparison of results with cannabis from public data bases.
Obtaining the NMR chemical profile of the cannabis sample involves obtaining the 1H and 13C spectrum.
The NMR technique allows the analysis of the content of secondary metabolites, mainly cannabinoids and terpenes.
Obtaining the NMR chemical profile of the cannabis sample involves the following steps: extraction, dissolution in deuterated solvent, obtaining and analyzing the 1H and 13C spectrum.
The sample extraction is performed with a solvent selected from among:
Additionally, the deuterated solvent is selected from the deuterated variants of: chloroform, acetone, methanol, benzene, heavy water, dimethyl sulfoxide, dichloromethane, trifluoroacetic acid, acetonitrile, pyridine, N,N-dimethylformamide and tetrahydrofuran, preferably chloroform.
Sample processing for NMR analysis involves the use of a chemical drying agent, selected from anhydrous magnesium sulfate, sodium sulfate and calcium chloride.
The determination can be performed based on the signal pattern in the 1H-NMR spectrum that appears between:
The integration ratio of the zones of the different aliphatic protons versus unsaturated ones is comprised in the range: 20:8 to 40:8, preferably 30:8, and corresponds to the chemical composition present in the extract.
In an additional embodiment, the 1H spectrum is confirmed by analysis of signals of 13C-NMR spectrum acquired from 0 to 180 ppm. There is a correspondence between signals corresponding to the aliphatic, unsaturated and aromatic carbon atoms with those found in 1H-NMR spectrum.
13C-NMR spectrum is analyzed in the range from 100 to 180 ppm, preferably the range from 160 to 180 ppm. In this area there are signals corresponding to carbon atoms assigned to carbonyl groups that belong to the terpenic fraction and which have not undergone decarboxylation in the extraction process, mainly due to the absence of very high temperatures in said stage.
13C-NMR spectrum analysis comprises at least 40 frequencies (ppm) of peaks associated with metabolites present in the sample, more preferably at least 60 frequencies.
The agreement between STR and NMR patterns obtained through the analyses, as well as their reproducibility, increases the degree of certainty in the identification of cannabis samples to values that have not been achieved so far. Until now, legal analysis of cannabis samples was generally limited to determining the net weight of the sample and analyzing the THC percentage, and therefore the combination of NMR and STR techniques has a clear advantage over the state of the art. It can be concluded that the combination of both methods allows a more accurate identification with less error than any other method known so far.
Samples can be from plants of the genus Cannabis and are selected from Cannabis sativa L. sativa and indica subspecies, sativa variety; Vavilov spontaneous variety; indica (Lam.) variety, kafiristanica (Vavilov) variety and combinations thereof, more preferably Cannabis sativa L.
The sample of the cannabis plant is selected from leaves, stem, bulb, seeds and flowers, preferably leaves.
The sample amount used to obtain the fingerprint is from 0.1 to 10 g, preferably 0.2 to 1.0 g, more preferably 0.5 g.
An additional object of the invention is the creation of a database comprising the fingerprints of samples of cannabis obtained by the method described above.
Such a database will enable the fingerprint of a sample to be compared with all previously obtained fingerprints stored in that database.
The present invention is illustrated by the following examples which are not intended to be limiting.
The analyses carried out have been made with leaf samples from 5 Cannabis sativa L varieties as shown in Table 2.
7 g × 1 Units
Molecular Analysis of DNA Markers
DNA Extraction
For the genetic sequencing analysis, the samples were kept frozen at a temperature of −20° C. from reception until the analysis. The sample is then cut into small portions using sterile blades and homogenized with liquid nitrogen. DNA extraction has been carried out using the method of cethylmethylammonium bromide (CTAB) in water on the samples, following a manual and standard forensic extraction method18. The main steps are described below:
The sample is ground with liquid nitrogen in a mortar to obtain a fine powder. Then, 300 μl of extraction buffer (0.1 mM EDTA, 10 mM Tris-HCl pH 7.5) with 0.2% beta-mercaptoethanol are added. The resulting product is mixed and incubated for 1 hour at 60° C. in a shaker. Then, 300 μl of isoamyl alcohol chloroform (24:1) are added and mixed by inversion. Centrifugation for 10 minutes at 14500 rpm is carried out and transfer of the aqueous phase into a clean tube, to which 1 volume (about 200 μl) of isopropanol is added, mixed by inversion and the samples are incubated for 10 minutes at room temperature. Then centrifugation of the tubes for 10 minutes at 10000 rpm is carried out, discarding the supernatant, washing the pellet obtained with 300 μl 70% ethanol and centrifugation of the tubes for 5 minutes at 10000 rpm, discarding the supernatant again and allowing the pellet to dry. Finally, the sample is redissolved in 50 μl of double-distilled, nuclease free water and the purified DNA is quantified.
DNA quantification and quality analysis (A280/230 and A230/A260) were obtained by a spectrophotometric method, using the NanoVue spectrophotometer. Each sample was analyzed in triplicate.
Sequencing the Rubisco Gene
The genetic confirmation of the species (Barcoding) was obtained by PCR amplification and Sanger sequencing of the Rubisco gene fragment. The sequence obtained was searched in the international GenBank database.
The amplified Rubisco gene fragment is the one delimited by the design of the primers published in the scientific article by Kress et al.21 The size of the amplification fragment generated with these primers is about 700 base pairs.
The PCR conditions were as follows: 98° C.-30 seconds, 40 cycles (98° C.-10 seconds; 52° C.-30 seconds; 72° C.-30 seconds), 72° C.-2 minutes.
The reagents, reaction volume and controls are as follows
Reagents kit used: New England Biolab (NEB) Q5 High-fidelity DNA polymerase M0491S.
The amplified gene fragment was analyzed in a 2% agarose gel.
For the analysis of the PCR results an agarose gel (2%) was prepared: Agarose D1 LE (Low EEOO) Cat. No E5000 INtRON biotechonology: 0.7 g. TBE 1× buffer: 35 ml. Sybr safe DNA gel stain. Invitrogen Ref S33102: 3,5 μl. The molecular weight marker used was the Omega M01-02 100 bp DNA ladder. Promega blue/Orange loading buffer 6× Ref: G190A was added to the samples before loading them into the gel. A volume of 3-5 μl of loaded PCR/marker product was loaded into each well. The electrophoresis conditions were 90 Watt for a period of 30-45 min.
A Sanger sequencing of the samples was additionally performed using the same primers19. The nucleotide sequences obtained were searched into the NCBI open access database, confirming that the amplified fragment corresponds to the Rubisco gene,
Analysis of STR Molecular Markers
A total of 13 pairs of previously published primers were used20, 21 for the amplification of the microsatellites detailed in table 4. For capillary electrophoresis, the Forward primer of each pair was marked with a fluorophore for subsequent separation and analysis21:
and for the analyses a PCR thermocycler, a gel documenter and a genetic analyser have been used. The genetic method has been developed by amplification of STRs microsatellite markers through multiplexed PCR, using the techniques previously indicated.
Multiplexed PCR (13 Markers):
PCR program: 98° C. 30 s; 7 cycles (98° C. 10 s; 61° C. 30 s, 72° C. 30 s); 5 cycles for each temperature (98° C. 10 s; touchdown* ° C., 30 s, 72° C. 30 s); 7 cycles (98° C. 10 s; 51° C. 30 s, 72° C. 30 s); 72° C. 2 min. Touchdown*: 60, 59, 57, 54, 52° C.
The concentration of primers in each reaction is shown in Table 5:
The reagents, reaction volume and controls are
Capillary Electrophoresis and Fragment Analysis
Fragment separation and detection of PCR products was performed in Genetic Analyzer 3130 (Applied Biosystems).
1-2 μL of each PCR product were loaded to a 10 μL mixture (9.5 μl of “Hi-Di Formamide®” and 0.5 μl of the LIZ® 500 molecular weight marker. The samples were denatured 5 minutes at 94° C. before being loaded into the Genetic Analyzer 3130, and run under the following conditions: Oven at 60° C.; prerun 15 kV; injection 1.2 kV, 16 s; run 15 kV, 1200 s; capillary length 36 cm; polymer: POP-7; Dye Set G5 probe set.
Simultaneously to the analysis of the fragments by capillary electrophoresis, a 2% agarose gel can be made according to the protocol indicated above, and part of the multiplex PCR product can be run therein to determine that the amplification occurred correctly.
The analysis of results was performed with GeneMapper® v5.0 Software (Applied Biosystems). They can be seen in
Table 7 shows the results obtained for the STR marker alleles (columns) in the samples tested (rows).
Repeated values have been found in the patterns of the markers and each of the markers can be assigned to the samples analyzed. Table 8 shows the number of identical STR markers in each comparison and table 9 shows the % similarity between the samples.
Although all samples have a different marker pattern, the degree of similarity between the 681 and 684 samples (8 identical markers, 66.67%) is so high that it cannot be said with sufficient certainty that these samples do not have the same origin. This is why the determination of phenotypic characteristics by means of the 1H and 13C spectra provides key information to obtain the fingerprint of each cannabis sample in order to determine its traceability.
Nuclear Magnetic Resonance
Optimization of the Extraction Step
In order to obtain the corresponding NMR spectra it is necessary to extract the metabolites present in the plant, a process that required previous optimization. For this purpose, a Soxhlet extraction equipment and different solvents (ethanol, isopropanol, dichloromethane, chloroform or hexane) were used for the extraction and at laboratory scale.
However, it was found that the extraction processes using a Soxhlet equipment, which in practice take place under heat and at temperatures close to the boiling point of the extraction solvent, lead to decarboxylation of cannabinoids into acidic forms. For this reason, it was decided to carry out the extraction at room temperature, thus preserving the acidic derivatives of cannabinoids. In this “cold” extraction (which includes values between 10 and 25° C.), the samples were previously lyophilized and, after being powdered, they were subjected to extraction with different solvents (ethanol, isopropanol, dichloromethane, chloroform, acetonitrile, tetrahydrofuran or hexane) during different times (8, 12, 24 hours). This study made it possible to establish the optimum conditions for the extraction by weight of the extract and the analysis of the cannabinoid content by means of a gas chromatography (GC) or liquid chromatography (HPLC) technique. The results are summarised in Table 10:
20%
17%
21%
19%
1percentage of grams of crude oil obtained after extraction from the dry plant
2percentage referred to the main cannabinoid content (THC + CBD) in grams per 100 g of extract measured by GC technique.
In view of the results obtained in the optimization of the extraction phase, it was found that extraction with ethanol at room temperature (25° C.) for 12 hours was as effective as extraction with heating and that it was the most efficient in terms of cannabinoid extraction performance compared to the use of other solvents for extraction. The establishment and definition of the optimal conditions of the extraction process was followed by a validation phase in which a set of three experiments were performed under such conditions, checking, after analysis of the results, the reproducibility and validity of the process.
The use of methanol as a solvent was also studied, obtaining values of percentage of extraction, temperature and time similar to ethanol. However, this compound was discarded due to its high toxicity.
As a conclusion, the best results were obtained with ethanol as an extraction solvent and during a 12-hour extraction period, as described in the experimental process below.
Analysis of Cannabis Samples by NMR
For NMR analysis, a sample of Cannabis sativa L. (0.5 g), previously freeze-dried and powdered, is extracted with absolute ethanol (15 mL) at room temperature, with stirring and for 12 hours. After this period, the extract is dried on a chemical drying agent, which is selected from one of the following: anhydrous sodium sulphate or anhydrous magnesium sulphate or anhydrous calcium chloride, filtered through a filter (pore size 0), and concentrated under vacuum drying in a rotary evaporator at 20° C. A sample of this extract (7 mg) is dissolved in deuterated chloroform (3 mL) and analyzed by NMR at room temperature. The analysis is done in duplicate, with a concentration of 15 mg of extract in 3 mL of deuterated chloroform. In all cases the sample is completely dissolved. Under these conditions and for plant samples, the technique has a detection limit of 25 μg/mL, a quantification limit of 75 μg/mL and an absolute error of 4%13.
The 1H-NMR-1H and 13C-NMR spectra were acquired in a 400 MHz instrument model ARX400 from Bruker. The residual solvent signal in CDCl3 (δ=7.24 ppm, 400 MHz, for 1H and 8=77.0 ppm, 100 MHz, for 13C) was used.
The five analyzed samples present a different pattern in the analyzed area, allowing to assign and correlate the signals found with each sample, finding peaks that are differentiating, which are those followed by an asterisk (“*”), obtained from
It can be seen that the pattern of chemical composition is very different between the 5 samples analyzed. Table 12 shows the number of signals identified in the 13C spectrum of each sample and how many of them were common to the other samples analyzed.
Thus, obtaining fingerprints by NMR analysis combined with the determination of STR genetic markers in cannabis samples allows to increase the degree of certainty in the identification thereof. Particularly in the case of samples whose genetic pattern is coincident (samples 681 and 684), the chemical composition pattern, obtained based on 13C-NMR, is different, confirming the possibility of differentiating samples based on their origin.
The absolute error considered for ppm allocation in each sample is less than 0.01%. The NMR analysis from the 1H and 13C spectrum of each sample has been performed in triplicate, showing to be reproducible.
Vargas, M. (2009). Drogas y Democracia: hacia un cambio de paradigma. Rio de Janeiro: Comisión Latinoamericana sobre Drogas y Democracia (pag. 7).
Brazilian marijuana by applying IRMS analysis to seized samples. Forensic Sci Int 160:35-43.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/ES2020/070248 | Apr 2020 | ES | national |
