RAPD和ISSR分子标记检测大麻的遗传多样性初探

目的利用随机扩增多态性DNA和简单序列重复区间扩增分子标记检测大麻遗传多样性,并探讨其在法医学中的应用价值。方法收集中国4省6个地区的100株大麻叶子样品,采用CTAB法提取基因组DNA,设计选择11个RAPD引物和13个ISSR引物,采用6%中性聚丙烯酰胺凝胶电泳-硝酸银染色法进行检测,根据出现的条带数目和片段大小等分析大麻的多样性。结果 11条RAPD引物扩增出的片段在200bp以上共52条,其中具有多态性的27条;ISSR引物扩增出126条,其中具有多态性的73条;多态性条带比率分别为51.9%和57.9%,其差异不具有统计学意义(P>0.05)。结论 RAPD和ISSR两种方法均可用于大麻遗传多样性分析,对检测毒品原植物的种类和来源地具有一定的应用前景。     

Use of Embryos Extracted from Individual Cannabis sativa Seeds for Genetic Studies and Forensic Applications

Legal limits on the psychoactive tetrahydrocannabinol (THC) content in Cannabis sativa plants have complicated genetic and forensic studies in this species. However, Cannabis seeds present very low THC levels. We developed a method for embryo extraction from seeds and an improved protocol for DNA extraction and tested this method in four hemp and six marijuana varieties. This embryo extraction method enabled the recovery of diploid embryos from individual seeds. An improved DNA extraction protocol (CTAB3) was used to obtain DNA from individual embryos at a concentration and quality similar to DNA extracted from leaves. DNA extracted from embryos was used for SSR molecular characterization in individuals from the 10 varieties. A unique molecular profile for each individual was obtained, and a clear differentiation between hemp and marijuana varieties was observed. The combined embryo extraction–DNA extraction methodology and the new highly polymorphic SSR markers facilitate genetic and forensic studies in Cannabis.     

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.     

Fatalities associated with fentanyl and co-administered cocaine or opiates

Fatalities associated with fentanyl hydrochloride are increasingly seen in Massachusetts. Between September 2005 and November 2006, 5009 medicolegal investigations associated 107 deaths with licit or illicit fentanyl use, along with a co-detection of an opiate/opioid or cocaine/benzoylecognine, or both. Deaths associated with illicit fentanyl use occur in younger people (39.4 vs. 61.5 years) with higher fentanyl (17.1 ng/mL vs. 4.4 ng/mL) and lower morphine (76.9 ng/mL vs. 284.2 ng/mL) postmortem blood concentrations, and more frequent cocaine co-intoxication (65% vs. 3%), than deaths associated with illicit fentanyl use. A wide range of postmortem blood concentrations of fentanyl was detected (trace-280 ng/mL), with a minimum concentration of 7 ng/mL of fentanyl strongly associated with illicit use of fentanyl in poly-drug cases. The most commonly detected opiates/opioids in illicit fentanyl users were: morphine (29%), oxycodone (14.5%), and methadone (14.5%). Ethanol, cannabinoids, diazepam, citalopram, and diphenhydramine were each detected in greater than 10% of the licit fentanyl cases. Most fentanyl abusers died at their own home and their deaths were most often classified as accidental. Mapping of primary residences of decedents revealed conspicuous clustering of the illicit fentanyl use cases, as opposed to the random pattern in licit use cases. Fentanyl misuse is a public health problem in Massachusetts.     

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.     

The frequency of alcohol,illicit and licit drug consumption in the general driving population in South-East Hungary

In the framework of the DRUID (Driving under the Influence of Drugs, Alcohol, and Medicines) EU-6 project, a roadside survey was performed in South-East Hungary to determine the incidence of alcohol and the most frequent illicit and licit drug consumption (amphetamines, THC, illicit and medical opiates, cocaine, ketamine, benzodiazepines, zopiclone and zolpidem) in the general driving population. All 3110 drivers stopped between 01 January 2008 and 31 December 2009 were checked for alcohol, and among them 2738 persons (87.7%) participated in the further examinations, on a voluntary basis. Licit and illicit drugs were determined from their oral fluid samples by GC–MS analysis.Illicit drugs were detected in 27 cases (0.99%), licit drugs in 85 cases (3.14%), and alcohol (cut off: 0.1 g/l) was found in 4 (0.13%) cases. Illicit drug consumption was the highest among men of the ages 18–34, during the spring, and on the week-end nights. With respect to licit drugs, the highest incidence was found among women over the age of 50, during the summer, and on the week-days. All alcohol positive cases were men over the age of 35. In comparison to international European averages, the alcohol and illicit drug consumption was low, but the licit drug consumption was over the European average.     

Growing cannabis with naphthalene in Rome

A young Italian male was investigated for possession of illicit marijuana in Rome. In his house, police found 80 cannabis plants, the plants were different sizes and located in a room with ultraviolet light, naphthalene, as a grey-white powder, was also found in his house. The man indicated that he used it for cannabis cultivation.     

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.     

A simple DNA extraction method for marijuana samples used in amplified fragment length polymorphism (AFLP) analysis

As a first step in developing a molecular method for the individualization of marijuana samples, we evaluated a plant DNA extraction kit. The QIAGEN plant DNeasy method uses a spin column format for recovery of DNA and is effective for obtaining high molecular weight DNA from leaf, flower (bud), and seed samples of marijuana. The average DNA yield was 125-500 ng per 100 milligrams of fresh plant tissue. The recovered DNA was of polymerase chain reaction (PCR) quality as measured by the ability to generate reproducible amplified fragment length polymorphism (AFLP) profiles. AFLP is a technique used to create a DNA profile for plant varieties and is being applied to marijuana samples by the authors to link growers and distributors of clonal material. The QIAGEN plant DNeasy method was simple, efficient, and reproducible for processing small quantities of marijuana into DNA.     

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.     

Salvia divinorum: an hallucinogenic mint which might become a new recreational drug in Switzerland

Salvia divinorum Epling & Jativa is an hallucinogenic mint traditionally used for curing and divination by the Mazatec Indians of Oaxaca, Mexico. Young people from Mexican cities were reported to smoke dried leaves of S. divinorum as a marijuana substitute. Recently, two S. divinorum specimens were seized in a large-scale illicit in-door and out-door hemp plantation. Salvinorin A also called divinorin A, a trans-neoclerodane diterpene, was identified in several organic solvent extracts by gas chromatography-mass spectrometry. The botanical identity of the plant was confirmed by comparing it to an authentic herbarium specimen. More plants were then discovered in Swiss horticulturists greenhouses. All these data taken together suggest that many attempts exist in Switzerland to use S. divinorum as a recreational drug. This phenomenon may be enhanced because neither the magic mint, nor its active compound are banned substances listed in the Swiss narcotic law.     

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.     

A survey of the potency of Japanese illicit cannabis in fiscal year 2010

In recent years, increased 'cannabis potency', or Δ(9)-tetrahydrocannabinol (THC) content in cannabis products, has been reported in many countries. A survey of Japanese illicit cannabis was conducted from April 2010 to March 2011. In Japan, all cannabis evidence is transferred to the Minister of Health, Labour and Welfare after criminal trials. The evidence was observed at Narcotics Control Department offices in major 11 cities. The total number of cannabis samples observed was 9072, of which 6376 were marijuana. The marijuana seizures were further classified, and it was found that in terms of the number of samples, 65.2% of them were seedless buds, and by weight 73.0% of them were seedless buds. Seedless buds were supposed to be 'sinsemilla', a potent class of marijuana. THC, cannabinol (CBN) and cannabidiol (CBD) in marijuana seizures exceeding 1g were quantified. The number of samples analyzed was 1115. Many of them were shown to contain CBN, an oxidative product from THC. This was a sign of long-term storage of the cannabis and of the degradation of THC. Relatively fresh cannabis, defined by a CBN/THC ratio of less than or equal to 0.1, was chosen for analysis. Fresh seedless buds (335 samples) contained an average of 11.2% and a maximum of 22.6% THC. These values are comparable to those of 'high potency cannabis' as defined in previous studies. Thus, this study shows that highly potent cannabis products are distributed in Japan as in other countries.     

A Capture–Recapture Model to Estimate the Size of Criminal Populations and the Risks of Detection in a Marijuana Cultivation Industry

Originally developed in biology, capture-recapture methodologies have increasingly been integrated into the study of human populations to provide estimates of the size of “hidden populations.” This paper explores the validity of one capture-recapture model—Zelterman’s (1988) truncated Poisson estimator—used to estimate the size of the marijuana cultivation industry in Quebec, Canada. The capture–recapture analysis draws on arrest data to estimate the number of marijuana growers “at risk of being arrested” for a period of five years (1998–2002). Estimates are provided for growers involved in two different techniques: (1) soil-based growing, and (2) hydroponics. In addition, the study develops an original method to estimate the prevalence of cultivation sites “at risk of detection.” A first set of findings shows that the cultivation industry is substantial; the estimated prevalence of growers compares to estimates of marijuana dealers in the province. Capture–recapture estimates are also used to compare the risks of being arrested for different types of offenders. Results indicate that hydroponic growers—those involved in large scale and sophisticated sites—face lower enforcement-related risks than growers involved in smaller enterprises. The significance of these findings is discussed in the context of the widespread development, both in Europe and in North America, of a successful domestic production-driven, rather than importation-driven, marijuana trade.

NEIGHBORHOOD DISORDER,INDIVIDUAL RELIGIOSITY,AND ADOLESCENT USE OF ILLICIT DRUGS: A TEST OF MULTILEVEL HYPOTHESES*

We hypothesize about the relationships among perceived neighborhood disorder, individual religiosity, and adolescent use of illicit drugs, marijuana and hard drugs; and the age‐varying effects of religiosity on illicit drug use. Applying hierarchical linear models to analyze the National Youth Survey data, we first find that neighborhood disorder and religiosity have hypothesized effects on illicit drug use independent of social bonding and social learning variables that partly mediate the effects. Second, religiosity buffers the effects of neighborhood disorder on illicit drug use. Third, the effects of religiosity on illicit drug use become stronger throughout adolescence. The implications of the findings are discussed.     

The relationship between marijuana use and intimate partner violence in a nationally representative, longitudinal sample

Intimate partner violence is a significant public health problem, as these behaviors have been associated with a number of negative health outcomes including illicit drug use, physical injury, chronic pain, sexually transmitted diseases, depression, and posttraumatic stress disorder. The current study examined the association between marijuana use and intimate partner violence using a longitudinal survey of adolescents and young adults ages 15 to 26 years. Data were obtained from 9,421 adolescents in the National Longitudinal Study of Adolescent Health (Add Health) Waves 1 through 4 (1995-2008). Marijuana use was measured in the past year at each wave and participants were categorized as "users" or "nonusers." Partner violence was constructed using six items (three pertaining to victimization and three concerning perpetration) from Wave 4 (2007-2008). Using these six items, participants were categorized as "victims only," "perpetrators only," or "victims and perpetrators." Survey multinomial regression was used to examine the relationship between marijuana use and intimate partner violence. Consistent use of marijuana during adolescence was most predictive of intimate partner violence (OR = 2.08, p < .001). Consistent marijuana use (OR = 1.85, p < .05) was related to an increased risk of intimate partner violence perpetration. Adolescent marijuana use, particularly consistent use throughout adolescence, is associated with perpetration or both perpetration of and victimization by intimate partner violence in early adulthood. These findings have implications for intimate partner violence prevention efforts, as marijuana use should be considered as a target of early intimate partner violence intervention and treatment programming.     

Insights for Contemporary Drug Policy: A Historical Account of Opium Control in India and Pakistan

Opium, as a tradable commodity, has a long history in the Indian sub-continent. This article offers a history of the production and distribution of both licit and illicit opium from 1773 to the present day in order to explore the lessons that the experiences of Indian and Pakistani can offer to contemporary drug policy. Four insights for contemporary drug control policy are developed from the historical analysis: (1) post-independence Pakistan and India illustrate the difficulties of controlling a regulated, licit, opium industry; (2) the relationship between Chinese and Indian opium production and exports may suggest that competition can be an effective impetus to production suppression; (3) developmental approaches to reducing production can limit the damages caused by opium suppression; (4) effective suppression requires alterations to institutional and structural conditions that facilitate production (i.e. reducing violent conflict, improving civil and criminal justice institutions efficiency or extending transport infrastructures).     

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.     

Marijuana Odor Perception: Studies Modeled from Probable Cause Cases

The 4th Amendment of the United States Constitution protects American citizens against unreasonable search and seizure without probable cause. Although law enforcement officials routinely rely solely on the sense of smell to justify probable cause when entering vehicles and dwellings to search for illicit drugs, the accuracy of their perception in this regard has rarely been questioned and, to our knowledge, never tested. In this paper, we present data from two empirical studies based upon actual legal cases in which the odor of marijuana was used as probable cause for search. In the first, we simulated a situation in which, during a routine traffic stop, the odor of packaged marijuana located in the trunk of an automobile was said to be detected through the driver's window. In the second, we investigated a report that marijuana odor was discernable from a considerable distance from the chimney effluence of diesel exhaust emanating from an illicit California grow room. Our findings suggest that the odor of marijuana was not reliably discernable by persons with an excellent sense of smell in either case. These studies are the first to examine the ability of humans to detect marijuana in simulated real-life situations encountered by law enforcement officials, and are particularly relevant to the issue of probable cause.