RESUMO
Cannabis sativa can be classified in two main types, according to psychotropic cannabinoid ∆9-tetrahydrocannabinol (∆9-THC) content: the drug-type and the fiber-type. According to the European Monitoring Center for Drugs and Drug Addiction, most of the European Union countries consider the possession of cannabis, for personal use, a minor offense with possibility of incarceration. Despite of the model of legal supply (i.e., Spanish cannabis clubs, Netherlands coffee shops) or medical use (i.e., Italy), cannabis remains the most used and trafficked illicit plant in the European Union. Differentiating cannabis crops or tracing the biogeographical origin is crucial for law enforcement purposes. Chloroplast DNA (cpDNA) markers may assist to determine biogeographic origin and to differentiate hemp from marijuana. This research aims: to identify and to evaluate nine C. sativa cpDNA polymorphic SNP sites to differentiate crop type and to provide information about its biogeographical origin. Five SNaPshot™ assays for nine chloroplast markers were developed and conducted in marijuana samples seized in Chile, the USA-Mexico border and Spain, and hemp samples grown in Spain and in Italy. The SNapShot™ assays were tested on 122 cannabis samples, which included 16 blind samples, and were able to differentiate marijuana crop type from hemp crop type in all samples. Using phylogenetic analysis, genetic differences were observed between marijuana and hemp samples. Moreover, principal component analysis (PCA) supported the relationship among hemp samples, as well as for USA-Mexico border, Spanish, and Chilean marijuana samples. Genetic differences between groups based on the biogeographical origin and their crop type were observed. Increasing the number of genetic markers, including the most recently studied ones, and expanding the sample database will provide more accurate information about crop differentiation and biogeographical origin.
Assuntos
Cannabis , DNA de Cloroplastos , Polimorfismo de Nucleotídeo Único , Cannabis/genética , Marcadores Genéticos , DNA de Cloroplastos/genética , México , Reação em Cadeia da Polimerase , Europa (Continente) , Itália , Chile , EspanhaRESUMO
Background: Cannabis plants and their seed have been used in many cultures as a source of medicine and feeding during history. Today, there is an increasing demand for cannabis seeds for medical use. Moreover, a seed sales market with no legal regulations has also grown. This may pose some issues if a quality control is not set in place. Identification of cannabis strains is important for quality control purposes in a nonregulated growing market and in cases of illegal traffic and medical use. Owing to the high price as a pharmacological drug, commercial products of cannabis plants and seeds for medical users are often subjected to adulterations, either when packing or distributing certified seeds in the market. Materials and Methods: Cannabis commercial seeds and cannabis seeds for medical use were analyzed with high-resolution melting (HRM) analysis using barcoding markers. Humulus lupulus L. plants from a local market were used as outgroup control. DNA barcoding uses specific regions of the genome to identify differences in the genetic sequence of conserved regions such as internal transcribed spacer (ITS) and rbcL. DNA barcoding data can be generated with real-time polymerase chain reaction combined with HRM analysis to distinguish specific conserved DNA regions of closely related species. HRM analysis is the method of choice for rapid analysis of sequence variation. Results: The melting temperature (Tm) of homogeneous packages was consistent with single genotypes. However, packages containing contaminating seeds showed Tm differences of 0.2°C on average. Conclusions: An effective, rapid, and low-cost method based on ITS nuclear DNA and on chloroplast rbcL regions for screening and detection of contamination in commercial cannabis seeds was developed and applied for the analysis of different samples. This approach can be used as a quality control tool for cannabis seeds or other plant material.
Assuntos
Cannabis , Código de Barras de DNA Taxonômico , Cannabis/genética , Cloroplastos/genética , Código de Barras de DNA Taxonômico/métodos , DNA Intergênico , DNA de Plantas/genética , Controle de Qualidade , Sementes/genéticaRESUMO
Cannabis sativa L. is a plant cultivated worldwide as a source of fiber, medicine, and intoxicant. Traditionally, C. sativa is divided into two main types: fiber type (hemp) and drug type. Drug-type C. sativa differs from hemp by the presence of a high quantity of the psychoactive drug, Δ9-tetrahydrocannabinol (Δ9 THC). Cannabis sativa is the most commonly used used illicit controlled substance in Chile. Chile is the third greatest consumer of Cannabis in South America. The objective of this study was to determine the genetic composition of ten drug seizures of Cannabis spp. in the south of Chile using a high resolution melting (HRM) strategy combined with a barcoding marker, ITS. C. sativa samples were selected from previously processed more than a thousand crime cases at the, Araucania region crime lab, National Dept. of Health. Ten cases were selected. Sample collection was based on the following: a) dry and fresh samples with no evidence of decomposition or degradation, b) defined plant fragments such as flowers and leaves from individual plants and, c) samples with different content of THC, CBN and CBD. Five sub samples were randomly selected from each case (N=50). The commercial Silver Haze strain was used as a control. Two real-time PCR and HRM analyses were conducted. The first analysis was performed with a representative sample of each of the 10 cases studied. Then a second assay was performed with all subsamples of cases 1, 5 and 8. Results showed that real-time PCR combined with HRM analysis using ITS allowed to determine the genetic composition of cannabis in all cases studied. The derivative of melting and the analysis of the shape of the curve and the peak of Tm, showed that three groups can be clearly distinguished. A first group exhibited a peak of Tm close to 87.4°C and includes cases 7 and 8. A second group had a peak of Tm close to 87.6°C and includes case 5. A third group displayed a peak of Tm close to 87.9°C and includes case 1, 6 and Silver Haze strain. A second experiment was performed using subsamples of cases 1, 5 and 8. Case 1 displayed a unique composition of the drug suggesting that this seizure contained cannabis clonally propagated. In case 5, two genotypes were present, therefore this could be associated with two strain or two different origin. Case 8, was composed of a mixture of cannabis strains indicating the presence of various crop type and/or different biogeographic origin. In general, our results suggested genetically homogeneous seizures from Araucanía Region. The high latitude (37° 35' and 39° 37' South latitude) and the natural geographic borders that surround southern Chile helps the control of cannabis traffic into the country. Finally, HRM analysis coupled with the barcode ITS demonstrated to be a rapid and low-cost screening method.
Assuntos
Cannabis/genética , Código de Barras de DNA Taxonômico , DNA Intergênico/genética , Chile , DNA de Plantas/genética , Tráfico de Drogas , Genética Forense/métodos , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Temperatura de TransiçãoRESUMO
In nature, there are >200 species of fungi with hallucinogenic properties. These fungi are classified as Psilocybe, Gymnopilus, and Panaeolus which contain active principles with hallucinogenic properties such as ibotenic acid, psilocybin, psilocin, or baeocystin. In Chile, fungi seizures are mainly of mature specimens or spores. However, clandestine laboratories have been found that process fungus samples at the mycelium stage. In this transient stage of growth (mycelium), traditional taxonomic identification is not feasible, making it necessary to develop a new method of study. Currently, DNA analysis is the only reliable method that can be used as an identification tool for the purposes of supporting evidence, due to the high variability of DNA between species. One way to identify the species of a distinctive DNA fragment is to study PCR products analyzed by real time PCR and sequencing. One of the most popular sequencing methods of forensic interest at the generic and intra-generic levels in plants is internal transcribed spacer (ITS). With real time PCR it is possible to distinguish PCR products by differential analysis of their melting temperature (Tm) curves. This paper describes morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory. The fungus species were identified using scanning electron microscopy (SEM), mass spectrometry, HRM analysis, and ITS sequencing. The sporological studies showed a generally smooth surface and oval shape, with maximum length 10.1⯵m and width 6.4⯵m. The alkaloid Psilocyn was identified by mass spectrometry, while HRM analysis and ITS sequencing identified the species as Psilocybe cubensis. A genetic match was confirmed between the HRM curves obtained from the mycelia (evidence) and biological tissue extracted from the fruiting bodies. Mycelia recovered from the evidence and fruiting bodies (control) were genetically indistinguishable.
Assuntos
Alucinógenos/análise , Micélio/genética , Psilocybe/classificação , Psilocibina/análogos & derivados , Chile , DNA Fúngico/análise , Tráfico de Drogas , Genética Forense , Cromatografia Gasosa-Espectrometria de Massas , Microscopia Eletrônica de Varredura , Psilocibina/análise , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise de Sequência de RNA , Esporos/genéticaRESUMO
The X-chromosomal short tandem repeats (X-STRs) DXS6800, DXS101 and DXS8377 were analysed in a population sample from Buenos Aires (Argentina) using a polymerase chain reaction (PCR) multiplex approach with fluorescent detection. We present allele frequencies for these loci in a population comprising 113 women and 99 men. The Hardy-Weinberg equilibrium (HWE) was tested in the female sample and no significant deviations were observed. The homogeneity of allele frequencies of men and women was compared by the Fisher's exact test and showed similar distributions. Linkage disequilibrium (LD) tests were performed in males for all pairs of loci and no significant associations were detected. Parameters of forensic interest were also estimated.
Assuntos
Cromossomos Humanos X , Frequência do Gene , Genética Populacional , Repetições de Microssatélites , Argentina , Impressões Digitais de DNA , Feminino , Loci Gênicos , Humanos , Masculino , Reação em Cadeia da Polimerase MultiplexRESUMO
DNA extracted from the fingernails of female victims of a violent or aggressive act may assist in the identification of the male. Sometimes with the current autosomal STR loci, however, the victim's profile may mask the perpetrator's DNA profile or the perpetrator's DNA may be substantially lower in quantity than that of the victim's DNA. Thus, under these conditions, no characterization is possible. In this paper, an alternative DNA extraction procedure was employed, and the application of an STR locus residing on the Y chromosome DYS19 was typed to allow for genetic characterization of the perpetrator in such cases.