Distribution of Chemical Phenotypes (Chemotypes) in European Agricultural Hemp (Cannabis sativa L.) Cultivars

In Europe, more than 50 approved cultivars of fiber hemp (Cannabis sativa L.) are in agricultural production. Their content of psychoactive tetrahydrocannabinol (THC) is legally restricted to <0.2% (%w/w in the dry, mature inflorescences). Cannabis strains with much higher THC contents are also grown, illegally or under license for drug production. Differentiation between these two groups relies on biochemical quantification of cannabinoid contents in mature floral material. For nonflowering material or tissue devoid of cannabinoids, the genetic prediction of the chemical phenotype (chemotype) provides a suitable method of distinction. Three discrete chemotypes, depending on the ratio of THC and the noneuphoric cannabidiol (CBD), can be distinguished: a “THC-predominant” type, a “CBD-predominant” type, and an intermediate chemotype. We present a systematic genetic prediction of chemotypes of 62 agricultural hemp cultivars grown in Europe. The survey reveals the presence of up to 35% B_T allele-carrying individuals (representing either a THC-predominant or an intermediate chemotype) in some cultivars—which is unexpected considering the legal THC limit of 0.2% THC. The fact that 100% of the seized drug-type seeds in this study revealed at least one B_T allele, reflects that plant breeding efforts have resulted in a fixation of the B_T allele in recreational Cannabis. To guarantee a sincere forensic application based on a genetic chemotype prediction, we recommend not to classify material of unknown origin if the samples size is below nine genetically independent individuals.     

Whole Plastome Sequences of Two Drug-Type Cannabis: Insights Into the Use of Plastid in Forensic Analyses

DNA is one of the fastest growing tools in forensic sciences, increasing reliability in forensic reports and judgments. The use of DNA has increased in different areas of the forensic sciences, such as investigation of plant species, where plastid DNA has been used to elucidate and generate evidence in cases of traceability of genetically modified and controlled plants. Even with several advances and the practice of using DNA in forensic investigations, there are just few studies related to the identification of genetic tools for the characterization of drug and nondrug-types of Cannabis. Herein, the whole plastomes of two drug-type Cannabis are presented and have their structures compared with other Cannabis plastomes deposited in the GenBank, focusing in the forensic use of plastome sequences. The plastomes of Cannabis sativa “Brazuka” and of the hybrid Cannabis AK Royal Automatic presented general structure that does not differs from the reported for other C. sativa cultivars. A phylogenomic analyses grouped C. sativa “Brazuka” with the nondrug C. sativa cultivars, while the hybrid Cannabis AK Royal Automatic placed isolated, basal to this group. This suggests that the analysis of plastomes is useful toward genetic identification of hybrids in relation to C. sativa.     

Validation of the 4‐aminophenol color test for the differentiation of marijuana‐type and hemp‐type cannabis

The analysis of cannabis plant material submitted to seized‐drug laboratories was significantly affected by the signing of the Agricultural Improvement Act of 2018, which defined hemp and removed it from the definition of marijuana in the Controlled Substances Act. As a result, field law enforcement personnel and forensic laboratories now are in need of implementing new protocols that can distinguish between marijuana‐type and hemp‐type cannabis. Colorimetric tests provide a cost‐effective and efficient manner to presumptively identify materials prior to submission to a laboratory for analysis. This work presents the validation of the 4‐aminophenol (4‐AP) color test and demonstrates its utility for discriminating between marijuana‐type and hemp‐type cannabis (i.e., typification). Validation studies included the testing of numerous cannabinoid reference materials, household herbs, previously characterized cannabis plant samples, and real‐case samples. The 4‐AP test reliably produces a pink result when the level of Δ⁹‐tetrahydrocannabinol (THC) is approximately three times lower than the level of cannabidiol (CBD). A blue result is generated when the level of THC is approximately three times higher than that of CBD. Inconclusive results are observed when the levels of THC and CBD are within a factor of three from each other, demonstrating the limitations of the test under those scenarios.     

Fast Discrimination of Marijuana using Automated High-throughput Cannabis Sample Preparation and Analysis by Gas Chromatography–Mass Spectrometry

In the United States, federal law and many state laws differentiate between marijuana and industrial hemp through delta-9-tetrahydrocannabinol (THC) levels, whereby the latter is defined as ≤0.3 percent THC on a dry weight basis. Many traditional cannabis identification methods employed by crime laboratories cannot accurately determine total THC quantities in accordance with federal and state regulations, or do so with increased time, labor, and risks of instrument damage. In order to quickly distinguish positive marijuana samples, a method was developed to identify plant material with a total THC level >1%. This novel, automated dispersive pipette extraction (DPX) method uses tip-based technology and an automated liquid handler to enable fast, hands-free selective isolation of THC and its precursors for downstream gas chromatography–mass spectrometry (GC-MS) analysis. The workflow proceeds with no repetitive manual effort and reduced need for instrument maintenance while enabling crime labs to legally identify marijuana through the detection of total THC above 1%. Recovery of THC using the DPX extraction method was 93% at 30 µg/mL and 78% at 500 µg/mL. Similarly, THCA-A recovery was 100% at 30 µg/mL and 74% at 500 µg/mL. Samples evaluated in a blind study (proficiency, hemp, and nonprobative case samples) were all accurately identified as greater than or less than 1% THC, with samples containing <1% THC being identified as “cannabis” and subjected to more discriminative analysis as needed.     

Evaluation of genetic markers for the analysis of THC levels of Cannabis sativa samples using principal component analysis – A preliminary study

Cannabis sativa is a worldwide commercial plant used for medicinal purposes, food and fiber production, and also as a recreational drug. Therefore, the identification and differentiation between legal and illegal C. sativa is of great importance for forensic investigations. In this study, principal component analysis (PCA), an exploratory data analysis technique, was tested to correlate the specific genotype with the concentration of tetrahydrocannabinol (THC) in the samples. C. sativa samples were obtained from legal growers in Piedmont, Italy, and from illegal drug seizures in the Turin region. DNA was extracted, quantified, amplified with a 13-loci multiplex STR and finally analyzed with an automated sequencer. The results showed a trend in the analyzed samples as they differed by their THC content and allele profiles. PCA yielded two clusters of samples that differed by specific allele profiles and THC concentrations. Further validation studies are needed, but this study could provide a new approach to forensic investigation and be a valuable aid to law enforcement in significant marijuana seizures or in tracing illicit drug trafficking routes.     

Characteristics of cannabinoids composition of Cannabis plants grown in Northern Thailand and its forensic application

The Thai government has recognized the possibility for legitimate cultivation of hemp. Further study of certain cannabinoid characteristics is necessary in establishing criteria for regulation of cannabis cultivation in Thailand. For this purpose, factors affecting characteristics of cannabinoids composition of Thai-grown cannabis were investigated. Plants were cultivated from seeds derived from the previous studies under the same conditions. 372 cannabis samples from landraces, three different trial fields and seized marijuana were collected. 100g of each sample was dried, ground and quantitatively analyzed for THC, CBD and CBN contents by GC-FID. The results showed that cannabis grown during March-June which had longer vegetative stages and longer photoperiod exposure, had higher cannabinoids contents than those grown in August. The male plants grown in trial fields had the range of THC contents from 0.722% to 0.848% d.w. and average THC/CBD ratio of 1.9. Cannabis in landraces at traditional harvest time of 75 days had a range of THC contents from 0.874% to 1.480% d.w. and an average THC/CBD ratio of 2.6. The THC contents and THC/CBD ratios of cannabis in second generation crops grown in the same growing season were found to be lower than those grown in the first generation, unless fairly high temperatures and a lesser amount of rainfall were present. The average THC content in seized fresh marijuana was 2.068% d.w. while THC/CBD ratios were between 12.6 and 84.09, which is 10-45 times greater than those of similar studied cannabis samples from the previous study. However, most Thai cannabis in landraces and in trial fields giving a low log(10) value of THC/CBD ratio at below 1 may be classified as intermediate type, whereas seized marijuana giving a higher log(10) value at above 1 could be classified as drug type. Therefore, the expanded information provided by the current study will assist in the development of criteria for regulation of hemp cultivation in Thailand.     

European validation of a Cannabis sativa 13-locus STR multiplex kit for genetic identification: A preliminary study

Cannabis sativa L. is a plant cultivated worldwide as a source of fiber, medicine and intoxicant. Traditionally, is divided into two main types: fiber type (hemp) and drug type (marijuana). Marijuana differs from hemp by the presence of a high quantity of the psychoactive drug, Δ9-tetrahydrocannabinol. The development of a validated method using short tandem repeats (STRs) could serve as an intelligence tool to link cases by means of genetic individualization or association of cannabis samples. For this purpose, a 13-locus STR multiplex method was developed, optimized, and validated by the Department of Forensic Science at Sam Houston State University (SHSU) according to relevant ISFG and SWGDAM guidelines. The European community considers C. sativa plants illegals, even though its consumption is accepted in precise and limited places (coffee shops or cannabis clubs in Netherlands and Spain). However, there are different gaps in the legislation of some European countries. For instance, in Italy, “weed” possession is decriminalized. Although trafficking and sale are prohibited, possession of small quantities of marijuana is considered only a civil offense. In order to proceed with the kit evaluation and inter-laboratory comparison, SHSU DNA laboratory sent blind cannabis DNA samples of known genotypes. Blind DNA samples were analyzed in different laboratories with different sequencers and analysis conditions. In this article, the goals were: a) to demonstrate that 13-locus STR kit for C. sativa is robust enough and reproducible, in all forensic laboratories, and b) to show the applicability of the STR system in association with Cannabis sativa cases for intelligence purposes to link multiple cases by means of genetic individualization or association of cannabis samples.     

利用压力循环仪提取骨骼DNA

目的探讨建立利用压力循环技术提取骨骼DNA的新方法。方法将11个不同部位骨骼样品分成3组,一组采用本实验室常规方法处理骨骼,一组采用压力循环仪处理骨骼,另外一组作为阴性对照,然后统一进行DNA的提取及定量。结果采用压力循环技术提取骨骼DNA时间比实验室常规方法缩短24小时以上,提取效率提高6%～49%。结论压力循环技术可以作为一种提取骨骼DNA提取的新方法之一。     

Simultaneous Identification of Four “Legal High” Plant Species in a Multiplex PCR High‐Resolution Melt Assay,

The international prevalence of “legal high” drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real‐time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high‐resolution melt using LCGreen Plus^®. The PCR assay was evaluated based on the following: (i) specificity and selectivity—primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity—serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability—sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging “legal high” species.     

人骨骼及牙齿DNA提取方法的比较和优化

目的人骨骼和牙齿DNA提取方法的比较和优化。方法收集18份不同个体的长骨、30颗磨牙和同一个体2根股骨、8颗磨牙。利用TissueLyser-Ⅱ组织破碎仪和PreFiler Express BTA^TM法医DNA提取试剂盒（BTA法）,应用Automate Express^TM自动化法医DNA提取系统提取DNA,进行STR分型,与脱钙法进行比较,并进行实验条件优化。结果用TissueLyser-Ⅱ结合BTA法,约2.5h即可完成骨骼和牙齿的DNA提取,分型成功率分别为94.4%和96.7%。与脱钙法比较,两种方法获得DNA质量浓度和检出率比较接近（P〈0.05）,但BTA法在操作过程方面更具优势。最佳样本量为100mg,消化时间为2h。结论采用TissueLyser-Ⅱ组织破碎仪结合BTA法对骨骼和牙齿进行DNA提取和分型检验,能满足实际检案的要求,可在法医学实践中选择使用。     

Automated extraction of DNA from reference samples from various types of biological materials on the Qiagen BioRobot EZ1 Workstation

We have validated and implemented a protocol for DNA extraction from various types of biological materials using a Qiagen BioRobot EZ1 Workstation. The sample materials included whole blood, blood from deceased, buccal cells on Omni swabs and FTA Cards, blood on FTA Cards and cotton swabs, and muscle biopsies. The DNA extraction was validated according to EN/ISO 17025 for the STR kits AmpF?STR^® Identifiler^® and AmpF?STR^® Yfiler^® (Applied Biosystems). Of 298 samples extracted, 11 (4%) did not yield acceptable results. In conclusion, we have demonstrated that extraction of DNA from various types of biological material can be performed quickly and without the use of hazardous chemicals, and that the DNA may be successfully STR typed according to the requirements of forensic genetic investigations accredited according to EN/ISO 17025.     

Genetic variation in hemp and marijuana (Cannabis sativa L.) according to amplified fragment length polymorphisms

Cannabis sativa L. (Cannabaceae) is one of the earliest known cultivated plants and is important in the global economy today as a licit and an illicit crop. Molecular markers distinguishing licit and illicit cultivars have forensic utility, but no direct comparison of hemp and marijuana amplified fragment length polymorphism (AFLP) has been made to date. Genetic variation was surveyed in three populations of fiber hemp and a potent cultivar of marijuana using AFLP markers. Ten primer pairs yielded 1206 bands, of which 88% were polymorphic. Eighteen bands represented fixed differences between all fiber populations and the drug cultivar. These markers have practical utility for (1) establishing conspiracy in the cultivation and distribution of marijuana, (2) identifying geographic sources of seized drugs, and (3) discriminating illegal, potent marijuana cultivars from hemp where the cultivation of industrial hemp is permitted.     

法医物证DNA自动化检验技术体系的研究

目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO100．4、75—2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,进行比较测试。在“全国公安机关DNA数据库应用系统”中建立并应用实验室信息管理系统（LIMS）模拟实施规范化DNA检案。结果1552份各类检材,采用工作站-磁珠法提取DNA效果最佳,STR检测成功率为95％,工作站-Chelex法为88％;二者分别与其手工提取法比较,成功率无明显差异。92个样本同期检测,自动化工作站较手工操作DNA检案时间可缩减1．25倍。结论工作站域珠法提取涉案检材DNA,可获得满意的STR分型结果。应用LIMS管控,可有效防控污染,明显提高检案效率及鉴定质量。     

Analysis of Δ-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography–electrospray ionization mass spectrometry

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for the confirmation of Δ⁹-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm³, 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC + H⁺], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d₃-THC + H⁺]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r² = 0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette^®. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette^® collection, 26 of the 40 cases had an undetectable THC.     

Determination of cannabinoids in biological samples using a new solid phase micro-extraction membrane and liquid chromatography-mass spectrometry

A simple, rapid and relatively solventless method for extraction of objective compounds would be useful for forensic, judicial and clinical purposes. Solid phase micro-extraction membrane (SPMEM) is one such extraction technique that integrates sampling, extraction and concentration into a single step, and combines the advantages of both the solid phase micro-extraction (SPME) and membrane separation. In this study, a new kind of membrane was prepared using polyamide and Tenax compounds, and applied to solid phase micro-extraction. Characteristics of the membrane such as adsorption capacity were tested. Extraction conditions such as adsorption time, desorption solvents, desorption time and assisted desorption treatment methods were studied and optimized. Tetrahydrocannabinol (THC) and cannabidiol (CBD) in blood and brain of the injected male mice, and in spiked human urine were extracted using this solid phase micro-extraction membrane method. The extracted THC and CBD were further determined with LC-MS using APCI. Ions analyzed in single ion monitoring mode were 315 for THC and CBD, and 318 for the deuterated THC internal standard.     

利用AutoMate Express~(TM)自动化法医DNA提取系统提取骨骼及牙齿DNA

目的探讨建立骨骼及牙齿DNA自动化提取的新方法。方法将33份骨骼及15份牙齿样本分别用冷冻研磨和手工处理两种方法研磨成粉,采用AutoMate ExpressTM自动化法医DNA提取系统提取DNA并定量。结果 AutoMate ExpressTM自动化法医DNA提取系统能够在3h左右完成骨骼、牙齿DNA的提取,两种方法处理的骨骼样本所得DNA质量浓度差异无统计学意义。冷冻研磨处理的骨骼和牙齿样本均获得了较好的STR分型结果,且牙齿样本所得DNA质量浓度高于手工提取所得。结论应用AutoMate ExpressTM自动化法医DNA提取系统是自动化提取骨骼、牙齿DNA的一种新方法,可应用于法医实际案件检验。     

A PCR marker Linked to a THCA synthase Polymorphism is a Reliable Tool to Discriminate Potentially THC‐Rich Plants of Cannabis sativa L.

Neither absolute THC content nor morphology allows the unequivocal discrimination of fiber cultivars and drug strains of Cannabis sativa L. unequivocally. However, the CBD/THC ratio remains constant throughout the plant's life cycle, is independent of environmental factors, and considered to be controlled by a single locus (B) with two codominant alleles (B_T and B_D). The homozygous B_T/B_T genotype underlies the THC‐predominant phenotype, B_D/B_D is CBD predominant, and an intermediate phenotype is induced by the heterozygous state (B_T/B_D). Using PCR‐based markers in two segregating populations, we proved that the THCA synthase gene represents the postulated B locus and that specific sequence polymorphisms are absolutely linked either to the THC‐predominant or the THC‐intermediate chemotype. The absolute linkage provides an excellent reliability of the marker signal in forensic casework. For validation, the species‐specific marker system was applied to a large number of casework samples and fiber hemp cultivars.     

Efficiency evaluation of a DNA extraction and purification protocol on archival formalin-fixed and paraffin-embedded tissue

Formalin-fixed and paraffin-embedded tissue (FF-PET) is an invaluable resource for retrospective molecular genetic studies, but the extraction of high-quality genomic DNA from FF-PET is still a problematic issue. Despite the range of DNA extraction methods currently in use, the association of phenol–chloroform extraction and silica-based purification protocols, reported in ancient DNA studies on archaeological bones, has, to our knowledge, not been used for DNA extraction from FF-PET yet. The present study compared the efficiency of three DNA extraction and purification protocols from two different FF-PET substrates, heart and liver, by using quantitative PCR and multiplex amplification.We showed that the method, using phenol–chloroform and the QIAamp DNA mini^® Kit (Qiagen), was the most effective DNA extraction and purification method and that the DNA quantity extracted from liver is statistically more important than that extracted from heart. Autosomal STR typing by multiplex amplifications gave partial allelic profiles with only small size products (less than 300 bases) amplified, suggesting that DNA extracted from FF-PET was degraded.In conclusion, the protocol presented here, previously described in studies on ancient bones, should find application in different molecular studies involving FF-PET material.     

Genotyping of DNA samples under adverse conditions of Low Copy Number—LCN (formolisados tissue samples and embedded in paraffin)

This paper aims to describe and evaluate a protocol for extraction of DNA (deoxyribonucleic acid) in formalinized tissues and embedded in paraffin for forensics genetic analysis. In outline the method is the removal of paraffin with an organic solvent in 0.3–0.5 mg of the sample of the tissue under study, followed by removal of formaldehyde, rehydration and soon after the extraction of genomic DNA. The extraction is achieved through the stages of cellular lysis, enzymatic digestion of proteins and DNA precipitation in ethanol medium. With the research we can conclude that even when the DNA is present in small quantities in conditions of extreme difficulties in its extraction, as formalinized tissues and embedded in paraffin, the technique of optimizing the extraction of DNA used both to organic extraction as Chelex, for use in the polymerase chain reaction (PCR), and possible the investigation of different samples of human tissue, biological samples, or was obtained under the conditions tested, a DNA with good quality and concentration. The samples were amplified for the mini-STRs loci using the product marketed in multilocus, using a methodology recommended by the supplier and validated for analysis of forensic DNA. Commercial kit was used MiniFiler from Applied Biosystems. The DNA fragments amplified by PCR showed that the extracted DNA had good amplification.     

Species Identification of Cannabis sativa Using Real‐Time Quantitative PCR (qPCR),

Most narcotics‐related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3′ exon‐trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real‐time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa.