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.     

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.     

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.     

First systematic evaluation of the potency of Cannabis sativa plants grown in Albania

Cannabis products (marijuana, hashish, cannabis oil) are the most frequently abused illegal substances worldwide. Delta-9-tetrahydrocannabinol (THC) is the main psychoactive component of Cannabis sativa plant, whereas cannabidiol (CBD) and cannabinol (CBN) are other major but no psychoactive constituents. Many studies have already been carried out on these compounds and chemical research was encouraged due to the legal implications concerning the misuse of marijuana. The aim of this study was to determine THC, CBD and CBN in a significant number of cannabis samples of Albanian origin, where cannabis is the most frequently used drug of abuse, in order to evaluate and classify them according to their cannabinoid composition. A GC-MS method was used, in order to assay cannabinoid content of hemp samples harvested at different maturation degree levels during the summer months and grown in different areas of Albania. This method can also be used for the determination of plant phenotype, the evaluation of psychoactive potency and the control of material quality. The highest cannabinoid concentrations were found in the flowers of cannabis. The THC concentrations in different locations of Albania ranged from 1.07 to 12.13%. The influence of environmental conditions on cannabinoid content is discussed. The cannabinoid content of cannabis plants were used for their profiling, and it was used for their classification, according to their geographical origin. The determined concentrations justify the fact that Albania is an area where cannabis is extensively cultivated for illegal purposes.     

Detection and identification of cannabis by DNA

The unambiguous identification of illicit substances, including Cannabis sativa, is a major concern of law enforcement agencies. Current methods of cannabis identification involve the use of techniques such as HPLC and GC to identify cannabinoids. A method for the identification of cannabis using DNA-specific primers has been developed and is described here. The nucleotide sequences between the trnL and trnF genes in the chloroplast of Cannabis sativa have been determined and Cannabis sativa-specific nucleotide sequences within the intergenic spacer between the trnL 3′ exon and trnF gene identified. Primers, made to these sequences, have been tested on a range of different plant extracts but only give a PCR product in the presence of Cannabis sativa. The successful production of a PCR product using these primers identifies the presence of cannabis.     

Investigations into the hypothesis of transgenic cannabis

The unusual concentration of cannabinoids recently found in marijuana samples submitted to the forensic laboratory for chemical analysis prompted an investigation into whether genetic modifications have been made to the DNA of Cannabis sativa L. to increase its potency. Traditional methods for the detection of genetically modified organisms (GMO) were used to analyze herbal cannabis preparations. Our analyses support the hypothesis that marijuana samples submitted to forensic laboratories and characterized by an abnormal level of Δ(9)-THC are the product of breeding selection rather than of transgenic modifications. Further, this research has shown a risk of false positive results associated with the poor quality of the seized samples and probably due to the contamination by other transgenic vegetable products. On the other hand, based on these data, a conclusive distinction between the hypothesis of GMO plant contamination and the other of genetic modification of cannabis cannot be made requiring further studies on comparative chemical and genetic analyses to find out an explanation for the recently detected increased potency of cannabis.     

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.     

RAPD analysis distinguishes Cannabis sativa samples from different sources

Samples of the seeds and seedlings of Cannabis sativa, and its dried leaves and flowerheads (marijuana), could be reliably distinguished by RAPD-PCR (Random Amplified Polymorphic DNA using the Polymerase Chain Reaction). DNA was best extracted from fresh tissues using buffers and the detergent cetyltrimethylammonium bromide; poorly dried tissue or inviable seed yielded coloured samples of degraded DNA. DNA was isolated from 51 C. sativa and two Humulus lupulus (hops) samples. Of the C. sativa samples 43 were from Australia (ten from Canberra gardens, eight from a New South Wales crop and 25 from two Queensland crops) and eight were from Papua-New Guinea (P-NG). A total of 102 different bands were obtained using four 10-nucleotide primers with arbitrarily chosen sequences. Banding patterns were compared by calculating pairwise distances using various algorithms, and presented using the neighbour-joining tree and multidimensional scaling methods. These showed a clear difference between C. sativa and H. lupulus, and separated the samples of the latter into three distinct groups; one group comprised all the P-NG samples, another the Canberra samples, and the third, the three crop samples.     

Evaluation of 19 short tandem repeat markers for individualization of Papaver somniferum

Papaver somniferum, commonly known as opium poppy, is the source of natural opiates, which are used as analgesics or as precursors in the creation of semi-synthetic opioids such as heroin. An increase in opioid addiction in the United States has resulted in high rates of illicit opioid use and overdoses. It has recently been shown that P. somniferum DNA suitable for genetic analysis can be recovered from heroin samples. The development of a comprehensive genetic individualization tool for opium poppy could serve to link cases and strengthen programs such as the Drug Enforcement Administration’s (DEA) Heroin Signature Program, which seeks to combat rising opioid use.The purpose of this study was to develop a quantitative real-time PCR (qPCR) method for the quantification of opium poppy DNA, compare three commercial DNA extraction kits for their ability to isolate DNA from poppy seeds, and evaluate nineteen opium poppy short tandem repeat (STR) markers for their use in a forensic identification panel. Such a panel could be used for individualizing samples and determining the geographic origin in heroin or poppy seed tea cases. The qPCR method was proven to be reproducible and reliable, specific for P. somniferum, and sensitive enough for forensic case-type samples. Of the three kits tested, the nexttec™ one-step DNA Isolation Kit for Plants was the optimal method and facilitated rapid extraction of DNA from poppy seeds. The majority of evaluated STR primer sets were unreliable or had low discriminatory power, limiting their use for individualization of poppy samples. A six-locus STR multiplex was developed and evaluated according to Scientific Working Group on DNA Analysis Methods (SWGDAM) and International Society of Forensic Genetics (ISFG) guidelines, including the use of a sequenced allelic ladder. The multiplex was found to have low discriminatory power, with greater than two-thirds of samples analyzed having just two different genotypes. The multiplex was determined to be unsuitable for individualization; however, a genotype map was developed as a proof of concept that these markers may be useful for determining the biogeographical origin of samples. Searching the poppy genome for new STR markers and developing new primer sets may be necessary for the creation of a powerful genetic tool for the individualization of P. somniferum.     

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.     

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.     

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.     

Development of STR profiles from firearms and fired cartridge cases

Fired cartridge cases are a common type of evidence found at crime scenes. However, due to the high chamber temperatures and touch nature of this evidence, DNA testing is not commonly sought because it is believed DNA is only present in low levels, whether it is due to initial low levels of DNA and/or DNA degradation from the heat or inhibition of the PCR reaction. Moreover, very few laboratories report STR typing success with fired cases. This study focused on obtaining STR profiles from fired cartridge cases using the AmpFℓSTR^® MiniFiler™ kit, which is designed to amplify DNA from low level, inhibited, and degraded samples. Comparisons to other STR amplification kits were also conducted. In attempt to simulate casework, random individuals loaded cartridges into a firearm. DNA was recovered from the fired cartridge cases using the double swab technique and extracted using an automated large volume DNA IQ™ method. Initially, testing focused on known shedders handling cartridges for 30 s prior to firing. A significantly greater number of alleles was obtained following amplification with the MiniFiler™ kit versus the PowerPlex^® 16 BIO kit. No alleles were observed using the Identifiler^® kit. In an attempt to better simulate casework, a random selection of laboratory personnel handled shotshells for as long as needed to load and fire the weapon. In this mock sample study, the MiniFiler™ kit successfully amplified an average of 22% of expected alleles from DNA recovered from shotshell cases versus the PowerPlex^® 16 BIO kit where an average of 7% of alleles were observed. However, the total number of alleles obtained from the two kits was not significantly different. The quality of the DNA obtained from fired cases was studied with evidence of inhibition in at least 11% of shotshell case samples. After swabbing the head and the hull of three shotshell cases separately, a significantly greater number of alleles was obtained from the hull as opposed to the head of the fired shotshell case. In addition, after firing, various internal firearm surfaces were swabbed, including the chamber of barrel, ejection port, and breechface, in an attempt to obtain amplifiable DNA. DNA was obtained from the chamber of the barrel and was amplifiable using the MiniFiler™ kit, although mixtures were obtained with extensive drop-in and drop-out making this analysis unlikely to aid an investigation.     

Utility validation of extraction of genomic DNA from hard tissues, bone and nail, using PrepFiler™ Forensic DNA Extraction Kit

STR profiling using hard tissues obtained from a severely decomposed body is sometimes a laborious work. There is now on a market a new DNA extraction kit, PrepFiler™ Forensic DNA Extraction Kit (AppliedBiosystems), and we tested it for missing persons. Postmortem intervals ranged from weeks to several years. Fifteen bone fragments and eleven nails were used in this report. Genomic DNA was quantified by QuantiFiler^® DUO Quantification Kit (AppliedBiosystems), and STRs were analyzed using AmpFlSTR^® Identifiler^® PCR Amplification Kit (AppliedBiosystems). The profiling of 16 STR loci was successful in all nail samples. However, STR profiling was successful in only 6 of 15 bone materials. Nine cases failed to analyze STR polymorphisms using another DNA extraction kit, the QIAamp DNA Mini Kit (QIAGEN). For bone samples, it seems that STR profiling depends on the quality of samples.     

Potency trends of delta9-THC and other cannabinoids in confiscated marijuana from 1980-1997

The analysis of 35,312 cannabis preparations confiscated in the USA over a period of 18 years for delta-9-tetrahydrocannabinol (delta9-THC) and other major cannabinoids is reported. Samples were identified as cannabis, hashish, or hash oil. Cannabis samples were further subdivided into marijuana (loose material, kilobricks and buds), sinsemilla, Thai sticks and ditchweed. The data showed that more than 82% of all confiscated samples were in the marijuana category for every year except 1980 (61%) and 1981 (75%). The potency (concentration of delta9-THC) of marijuana samples rose from less than 1.5% in 1980 to approximately 3.3% in 1983 and 1984, then fluctuated around 3% till 1992. Since 1992, the potency of confiscated marijuana samples has continuously risen, going from 3.1% in 1992 to 4.2% in 1997. The average concentration of delta9-THC in all cannabis samples showed a gradual rise from 3% in 1991 to 4.47% in 1997. Hashish and hash oil, on the other hand, showed no specific potency trends. Other major cannabinoids [cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC)] showed no significant change in their concentration over the years.     

Field testing of collection cards for Cannabis sativa samples with a single hexanucleotide DNA marker

The validity and feasibility of using DNA collection cards in the field for preservation and analysis of Cannabis sativa genotypes were investigated using a highly specific hexanucleotide marker. Collection cards were submitted to the National Marijuana Initiative, which selectively trained and managed the collection of specific types of samples from a variety of participating agencies. Samples collected at seizure sites included fresh marijuana leaf samples, dried "dispensary" samples, U.S. border seizures, and hashish. Using a standardized PCR kit with custom-labeled oligonucleotide primers specific to marijuana, collection cards produced eight genotypes and 13 different alleles, extremely low baselines, and no cross-reactivity with control plant species. Results were produced from all sample types with the exception of hashish. Plant DNA collection cards represent an easily implementable method for the genetic identification and relatedness of C. sativa street and grow site-seized samples with applications for databasing and market disruption.     

05式警用转轮手枪弹壳接触DNA检验方法的比较

目的比较05式警用转轮手枪弹壳表面接触性DNA检验方法,为实际检验提供参考和借鉴。方法制备40例击发后手枪弹壳的模拟样本,分别用两步转移法提取弹壳表面不同部位检材,采用两种DNA提取法和两种扩增试剂盒对样本进行STR分型检验,比较评价检验结果。结果避开发射药残留区域采用两步转移法提取样本,有助于提高检出率;Chelex-100联合Microcon-100法提取模板DNA的产量最高可达1.18ng,高于Mini M48试剂盒法(0.91ng);MiniFilerTM试剂盒的等位基因检出率(23.61%)高于IdentifilerTM试剂盒(6.41%)。结论采用选择适当区域提取检材,采用Chelex-100联合Microcon-100法提取DNA,经MiniFilerTM试剂盒扩增,进行弹壳接触DNA分型的效果较好。     

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.     

Characterization of the polymorphic repeat sequence within the rDNA IGS of Cannabis sativa

The rDNA intergenic spacer (IGS) structure of Cannabis sativa contains six variable repeat motifs within a locus spanning 1387 base pairs. The degree of variation of the first three motifs was examined using 77 samples from cannabis samples. The samples originated from five seizures in Taiwan and seed stocks from six different countries. The results showed that there were four types of sequences producing PCR products at either 255, 260, 264 or 265 base pairs. The data obtained indicates that this region of rDNA IGS exhibits a degree of polymorphism that while insufficient by itself can be added to a multiplex with other cannabis STR loci.     

A highly polymorphic STR locus in Cannabis sativa

We report on the first short tandem repeat (STR) locus to be isolated from the plant Cannabis sativa. The STR locus, isolated by a hybrid-capture enrichment procedure, was found to contain a simple sequence repeat motif of 6 bp. This 6 bp repeat motif showed no variation in repeat length but with minor variations in repeat unit sequences. The data show the locus to be highly polymorphic with the number of repeat units ranging from 3 to 40 in 108 screened samples. The observed heterozygosity was approximately 87.04%. The forward and reverse primers (CS1F and CS1R) produced no PCR products in cross-reaction study from 20 species of plants, including highly related species such as Humulus japonicus and Nicotiana tabacum. This hexanucleotide repeat DNA locus could be used to identify cannabis samples and predict their genetic relationship as the test is specific to C. sativa and is highly reproducible.