Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay

Cannabis sativa is both an illegal drug and a legitimate crop. The differentiation of illegal drug Cannabis from non-drug forms of Cannabis is relevant in the context of the growth of fibre and seed oil varieties of Cannabis for commercial purposes. This differentiation is currently determined based on the levels of tetrahydrocannabinol (THC) in adult plants. DNA based methods have the potential to assay Cannabis material unsuitable for analysis using conventional means including seeds, pollen and severely degraded material. The purpose of this research was to develop a single nucleotide polymorphism (SNP) assay for the differentiation of "drug" and "non-drug"Cannabis plants. An assay was developed based on four polymorphisms within a 399 bp fragment of the tetrahydrocannabinolic acid (THCA) synthase gene, utilising the snapshot multiplex kit. This SNP assay was tested on 94 Cannabis plants, which included 10 blind samples, and was able to differentiate between "drug" and "non-drug"Cannabis in all cases, while also differentiating between Cannabis and other species. Non-drug plants were found to be homozygous at the four sites assayed while drug Cannabis plants were either homozygous or heterozygous.     

Delta(9)-THC, 11-OH-Delta(9)-THC and Delta(9)-THCCOOH plasma or serum to whole blood concentrations distribution ratios in blood samples taken from living and dead people.

The recreational use and abuse of Cannabis is continuously increasing in Switzerland. Cannabinoids are very often detected alone or in combination with other drugs in biological samples taken from drivers suspected of driving under the influence of drugs. Moreover, they are also frequently found in blood specimens from people involved in various medico-legal events, e.g. muggings, murders, rapes and working accidents as well. In order to assess the influence of Cannabis exposure on man behavior and performances, it is often needed to estimate the time of Cannabis use. For that purpose two mathematical models have been set up by Huestis and coworkers. These models are based on cannabinoids concentrations in plasma. Because plasma samples are rarely available for forensic determinations in our laboratory, it could be useful to assess the time-laps since Cannabis use through these models from whole blood values. One prerequisite to the use of these models from whole blood values is the knowledge of the plasma to whole blood concentrations distribution ratios of cannabinoids. In this respect, the Delta(9)-THC, 11-OH-Delta(9)-THC and Delta(9)-THCCOOH concentrations were measured in plasma and whole blood taken from eight volunteers who smoke Cannabis on a regular basis. Cannabinoids levels were also determined in "serum" and whole blood samples taken from six corpses. The values of the plasma to whole blood distribution ratios were found to be very similar and their individual coefficient of variation relatively low suggesting that plasma levels could be calculated from whole blood concentrations taken into account a multiplying factor of 1.6. The data obtained postmortem suggest that the distribution of cannabinoids between whole blood and "serum" is scattered over a larger range of values than those determined from living people and that more cannabinoids (mean value of the serum/whole blood concentrations ratios=2.4) can be recovered from the "serum" fraction. The successful use of the mathematical models of Huestis and coworkers may, therefore, rely in part upon the selection of the appropriate blood sample, i.e. plasma. When plasma is not available, whole blood values could be considered with some caution taken into account a multiplying factor of 1.6 to calculate plasma concentrations from blood values. In the case of blood samples taken after death, the use of these models to assess the time of Cannabis use is not recommended.     

AFLP技术鉴别罂粟、虞美人和大麻种属差异的初步研究

目的探讨采用AFLP技术检测植物DNA,鉴别罂粟、虞美人和大麻植物种属间差异的方法。方法收集罂粟根、茎、叶、花、果以及虞美人和大麻叶检材,用AxyPrep DNA试剂盒提DNA,经EcoRI/MseI酶切,人工接头及PCR预扩增,用E-ACA/M-CAG、E-ACT/M-CTC、E-ACC/M-CTA、E-ACC/M-CTG、E-AGC/M-CTT、E-AGG/M-CTA6对标记了荧光的选择性引物进行PCR扩增,其产物在的CEQ8000遗传分析仪上检测。结果6对引物分别在罂粟、大麻和虞美人样本中检出27~46、5~20、4~31条扩增片段,种属间存在明显差异;同一罂粟根、茎、叶、花和果的DNA检测结果相同。结论罂粟、虞美人和大麻3种植物的AFLP分析结果显示出的差异性,同一植株不同部位DNA AFLP结果的同一性,有可能用于检测未知植物检材的种属来源。     

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.     

Genetic variation of 23 STR loci in a Northeast Colombian population (department of Santander)

The most efficient markers to solve filiation cases are the STRs, including complex cases that require the analysis of a greater number of markers. In this study samples from 123 unrelated individuals from the department of Santander (northeast Colombia) were typed for 23 autosomal STRs included in VeriFiler express kit (Thermo Fisher Scientific),and their allele frequencies and parameters of forensic relevance were determined. Results demonstrate independence within and between the loci analyzed, and the accumulate power of exclusion for the full set of markers was high (99.9996%), as well as the match probability, which was 1 in 8.77E + 29. Therefore, this northeast Colombian population database can be used in forensic to estimate the frequency of the genetic profile using of a multiple locus including in this DNA kit.     

Application of HRM-PCR (high resolution melting PCR) for identification of forensically important Coleoptera species

Precise estimation time of death is one of key task of forensic entomology. Especially interesting is Coleofauna present at all stages of cadaver decomposition. The morphological identification of Coleoptera species from varying life stages to species level is time-consuming and needs highly qualified entomologists. Among different molecular methods of species identification very promising is high-resolution melting PCR. It allows fast single-tube assignment of analyzed sample to species based on amplicon melting profile. The object of this study were different specimens of Coleoptera collected at pig cadavers in Łomna (central Poland) during 2012 - 2014. Specimes were identified to species by experts of corresponding Coleoptera families. From 120 collected specimens belonging to four families and twelve species HRM-PCR correctly identified specimens belonging to three families and eight species.     

Preliminary data on the usefulness of internal transcribed spacer I (ITS1) sequence in Cannabis sativa L. identification.

A method is described to identify an unknown sample of plant material of forensic interest as Cannabis sativa L. The method consists in comparing the sequence of the nuclear ribosomal DNA Internal Transcribed Spacer I (ITS1) of the unknown sample with a Cannabis sequence. Our preliminary results show that the ITS1 is an ideal molecule for the identification of a sample suspected to be marijuana.     

A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples

In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drug-type from the fibre-type varieties. A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency plants, we chose the ΔΔCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished. As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested.     

The detection and persistence of Cannabis sativa DNA on skin

The presence of Cannabis sativa DNA was detected on the skin of persons who have recently handled both leaf and resinous material. The persistence of C. sativa DNA was examined on the skin. The subjects were asked to either repeatedly rub their hands on their trousers, place their hands repeatedly into their pockets or wash their hands in soap and water. After rubbing the hands on trousers or placing them in pockets C. sativa DNA could still be detected. No DNA could be detected after washing the hands.     

男性性功能鉴定（附73例报告）

报道７３例男性性功能鉴定，对目前常用的检测方法，手淫法、血管活性药物海绵体内注射作出评价，并认为对手淫法能勃起者与海绵体内注射罂粟碱不能勃起者，有认定价值。性心理调查、配偶方接受检验对勃起功能障碍的类型有鉴别作用。     

Development of the Investigator STR GO! Kits for the direct amplification of reference samples

STR analysis of forensic reference samples can be performed using a novel direct amplification method. Two assays have been developed for this purpose. One covers the CODIS set of markers and the other covers the ESS including SE33. The method gives balanced DNA profiles with high first pass rates for buccal swabs and blood and buccal cells on FTA paper. The addition of SNP primers compensates for profile imbalances caused by three known binding site mutations.     

Application of mtDNA SNP analysis in forensic casework

In this study six forensic cases are presented where the routine analysis of samples for short tandem repeats (STRs) failed. The sequencing of the mitochondrial hypervariable region I (HVR I) also failed. Nevertheless, it was possible to analyse the samples with mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) via SNaPshot technique. The age of the analysed samples ranged from 2 months to 1400 years. Saliva-, blood-, sperm-, hair-, tooth- and bone-samples were investigated. Furthermore the mtDNA SNP analysis of a forensic case sample showing a mixed stain profile is presented. It was possible to discriminate two different haplogroups in this mixed-person stain. If compared to another mtDNA SNP profile that was found in a hair, the discriminating SNPs of the hair were as well found in the mixed-person stain.To disburden the SNP analysis in forensic casework, haplogroup assignment criteria and quality criteria for mtDNA SNaPshot analysis are announced.     

On the legality of cannabis; the responsibility of the expert witness.

The controversy concerning the taxonomic status of the genus Cannabis has now advanced to a stage where the forensic scientist has limitations to his testimony in identification of "marihuana" plant material in jurisdictions where the law defines "marihuana" as Cannabis sativa L. Whether the genus Cannabis is monotypic or polytypic is as yet uncertain, but recent taxonomic reviews weigh heavily toward the existence of three or more species within the genus. The taxonomists or forensic scientists cannot, therefore, positively state for fact that C. sativa is the only species existing within the genus Cannabis. The popular concept of "marihuana" is actually based on the chemical characteristics of the plant Cannabis, rather than on the taxonomic classification. This is evident in its inclusion as a drug or hallucinogenic substance under Federal and local statutes. It is therefore proposed that "marihuana" be redefined legally to include all members belonging to the genus, in jurisdictions where legal definition warrants such an act, or that these jurisdictions follow the format set forth by Federal rulings.     

Validation studies of rapid stain identification-blood (RSID-blood) kit in forensic caseworks

When bloodstains are detected at crime scene using presumptive tests (e.g. luminol, phenolphthalein, leuchomalachite green), it is important to establish the real human nature of each stain. This is possible using confirmatory tests. One of these is rapid stain identification-blood (RISD-blood) a lateral flow immuno-chromatographic strip test format which allows the identification of human blood by detection of glycophorin A, a red blood cell membrane antigen, using two anti-human glycophorin A (GPA) monoclonal antibodies.The aim of this study is to assess the sensitivity of RSID-blood test in old, degraded bloodstains and in some bloodstains previously treated with BlueStar Forensic, a presumptive test which is often used in crime scene investigations to detect latent bloodstains. The genetic analysis of all bloodstains of confirmed human nature was subsequently performed using the AmpF1STR Identifiler PCR Amplification Kit (Applied Biosystems), to validate the possibility of obtain a consistent and reliable DNA typing results.     

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.     

How to regulate forensic familial DNA searching in Hungary?

Forensic DNA analysis has the potential to provide useful information for criminal justice even in cases where there is no match, neither between the DNA profile generated from the crime scene and the existing DNA profiles in criminal databases, nor between the DNA collected at a crime scene and potential suspects. In contrast to traditional forensic genetic testing, forensic familial DNA searching does not provide evidence, but helps to generate investigative leads and narrow down the range of potential offenders. The aim of this study is to examine, whether there is a need for special regulation of this topic in Hungary.     

Rapid genomic DNA extraction (RGDE)

Hyperpolymorphic short tandem repetitive DNA sequences, STRs or microsatellites, have become widely used in human identification, particularly in criminal cases and in mass disasters. In such cases the substrates for the analyses may be decomposed as a biological material, a fact that has to be taken into account when choosing the appropriate casework methods. Nowadays expanded windows have been opened to the world especially in the area of genetic and biology science by performance of big projects such as human genome project. In this regard, one of the primary and important steps for all is DNA extraction with high quality and quantity in minimum time from biological material. By using RGDE method, genomic DNA with high quality and quantity can be acquired in the shortest time which has been presented in the world up to now. In this paper we report the evaluation of DNA extraction in this method.     

Deoxyribonucleic acid (DNA) analysis by restriction fragment length polymorphisms of blood and other body fluid stains subjected to contamination and environmental insults.

Deoxyribonucleic acid (DNA) restriction fragment length polymorphism (RFLP) profile results were obtained from bloodstains and other body fluid stains subjected to mixture with other body fluids, environmental insults (sunlight and temperature), different substrates (cotton, nylon, blue denim, glass, aluminum, and wood), and contaminants (gasoline, bleach, sodium hydroxide, soil, motor oil, detergent, phosphate salt, glacial acetic acid, and microorganisms). Of the samples that produced profile results, all had profiles that were consistent with those of untreated control samples.     

The collapse of an Italian cemetery into the sea: Forensic approach to human remains identification

On February 22nd, 2021, a landslide on the Italian coast caused the collapse of an old cemetery. About 370 coffins tumbled and more than 200 fell into the sea. 333 groups of unidentified human remains were found: 140 decomposed bodies and 193 bags of commingled skeletal remains. The Medical Staff of Legal and Forensic Medicine was involved for analyzing the remains in order to identify and bury them. The remains involved belonged to people who died between the end of the XIX century and 2017; all were interesting by advanced transformative phenomena. For the identifications, new forms, based on the Interpol DVI ones, were created. Information was collected by relatives through a specific antemortem form. Relatives’ information and post-mortem data were compared: 19 body were identified thanks to secondary methods (like object in the bury, dresses, medical devices). 147 bone samples (long bones and teeth) were collected for the genetic analysis. Among the 77 relatives eligible for a genetic comparison, 66 gave consent to DNA swab for collection and genetic typing. Currently, after 48 samples DNA analysis (STRs and Y-polymorphism) 12 remains were identified, 21 presented a profile suitable for comparison but without attribution, and 7 did not return a comparable profile caused by stochastic effects. 31 subjects have been identified and the genetics analysis are still in progress. The Cemetery collapse shows that every disaster requires a tailored approach.     

Cannabinoids in blood and urine after passive inhalation of Cannabis smoke

To test the possibility that cannabinoids are detectable following passive inhalation of Cannabis smoke the following study was performed. Five healthy volunteers who had previously never used Cannabis, passively inhaled Cannabis smoke for 30 min. Cannabis smoke was provided by other subjects smoking either marijuana or hashish cigarettes in a small closed car, containing approximately 1650 L of air. delta 9-Tetrahydrocannabinol (THC) could be detected in the blood of all passive smokers immediately after exposure in concentrations ranging from 1.3 to 6.3 ng/mL. At the same time total blood cannabinoid levels (assayed by radioimmunoassay [RIA] ) were higher than 13 ng/mL in four of the volunteers. Both THC and cannabinoid blood concentrations fell close to the cutoff limits of the respective assays during the following 2 h. Passive inhalation also resulted in the detection of cannabinoids in the urine by RIA and enzyme multiple immunoassay technique (EMIT) assays (above 13 and 20 ng/mL, respectively). It is concluded that the demonstration of cannabinoids in blood or urine is no unequivocal proof of active Cannabis smoking.