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.     

云南大麻DNA的提取及检测初步研究

目的探讨云南大麻DNA的提取及检测方法。方法运用改进的SDS微量法提取大麻总DNA,对所得DNA进行PCR检测。结果用所筛选到的大麻引物检测了云南大麻的DNA遗传标记特征,图谱清晰,结果稳定。结论本方法能有效提取并检测大麻DNA,为实现大麻的品种鉴定、遗传多态性研究及来源追踪提供有价值的科学依据。     

The Identification of Ingested Dandelion Juice in Gastric Contents of a Deceased Person by Direct Sequencing and GC–MS Methods*

Abstract: DNA and chemical analysis of gastric contents of a deceased person were handled in this work. The body of the victim was discovered in his car, submerged in a lake. We were asked to determine whether or not the gastric contents of the victim harbored drugs and dandelion material. It was suspected that the victim had been murdered by poisoning with an excess amount of sleeping medication (doxylamine), which had been homogenized with dandelion. The concentrations of 11.4 and 27.5 mg/kg of doxylamine detected from spleen and liver of the victim were far higher than the assumed therapeutic concentration. Via gas chromatography–mass spectrometry (GC–MS) analysis and direct sequencing analysis of plant genetic markers such as intergenic transcribed spacer, 18S ribosomal RNA (rRNA), rbcL and trnLF, it was confirmed that the gastric contents of the victim contained taraxasterol, which is one of the marker compounds for dandelion and contained dandelion species‐specific rbcL and trnL‐trnF IGS (trnLF) sequences. The initial PCR of the genomic DNA isolated from the gastric contents showed insufficient quantity, and the second PCR, of which the template was a portion of the initial PCR products, exhibited a sufficient quantity for direct sequencing. rbcL and trnLF located in the cpDNA resulted in the successful determination of dandelion DNA in a decedent’s stomach contents. GC–MS identifies the actual presence of a taraxasterol at 28.4 min. Raw dandelion was assumed to be used as a masking vehicle for excess sleeping drug (doxylamine).     

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.     

An Assessment of the Utility of Universal and Specific Genetic Markers for Opium Poppy Identification

Abstract: The proper identification of illicit plants such as Papaver somniferum L (opium poppy) is important for law enforcement agencies. The identification of opium poppy was presently tested using 10 genetic markers that are universal for all plants or specific to a few poppy plants. The genetic distances of universal markers such as nuclear internal transcribed spacer (ITS), 18S rRNA, plastid rbcL, and trnL‐trnF intergenic spacer (IGS) of 14 species included in the Papaveraceae and Fumariaceae family were acquired by sequence comparisons. Both the ITS region and trnL‐trnF IGS showed high levels of interspecific divergence. Six Papaver genera‐specific markers were developed from coding regions involved in morphine biosynthesis. Three markers (TYDC, NCS, and BBE) produced amplicons only in opium poppy, providing a presence/absence test for opium poppy, while three additional markers (CYP80B1, SAT, and COR) were genus specific. These 10 markers might be useful for the forensic DNA analysis of opium poppy.     

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.     

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.     

DNA polymorphisms in the tetrahydrocannabinolic acid (THCA) synthase gene in "drug-type" and "fiber-type" Cannabis sativa L

The cannabinoid content of 13 different strains of cannabis plant (Cannabis sativa L.) was analyzed. Six strains fell into the "drug-type" class, with high Delta-9-tetrahydrocannabinolic acid (THCA) content, and seven strains into the "fiber-type" class, with low THCA using HPLC analysis. Genomic DNA sequence polymorphisms in the THCA synthase gene from each strain were studied. A single PCR fragment of the THCA synthase gene was detected from six strains of "drug-type" plants. We could also detect the fragment from seven strains of "fiber-type" plants, although no or very low content of THCA were detected in these samples. These were 1638 bp from all 13 strains and no intron among the sequences obtained. There were two variants of the THCA synthase gene in the "drug-type" and "fiber-type" cannabis plants, respectively. Thirty-seven major substitutions were detected in the alignment of the deduced amino acid sequences from these variants. Furthermore, we identified a specific PCR marker for the THCA synthase gene for the "drug-type" strains. This PCR marker was not detected in the "fiber-type" strains.     

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.     

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.     

Detection and Identification of Kratom (Mitragyna speciosa) and Marijuana (Cannabis sativa) by a Real-Time Polymerase Chain Reaction High-Resolution Melt Duplex Assay,

Mitragyna speciosa (MS), a plant commonly known as kratom, is a widely used “legal high” opiate alternative for pain relief. DNA extracted from MS and 26 additional plant species was amplified by PCR using primers targeting the strictosidine beta-D-glucosidase (SGD) and secologanin synthase 2 (SLS2) genes and detected by high-resolution melt curves using three intercalating dyes. Amplicon sizes were confirmed using agarose gel electrophoresis. The observed melt temperatures for SGD and SLS2 were 77.08 ± 0.38°C and 77.61 ± 0.46°C, respectively, using SYBR^® Green I; 80.18 ± 0.27°C and 80.59 ± 0.08°C, respectively, using Radiant^™ Green; and 82.19 ± 0.04°C and 82.62 ± 0.13°C, respectively, using the LCGreen^® PLUS dye. The SLS2 primers demonstrated higher specificity and identified MS DNA at 0.05 ng/μL. In a duplex reaction, SLS2 and tetrahydrocannabinoic acid synthase gene primers detected and differentiated MS and Cannabis sativa (CS) by melt peaks at 82.63 ± 0.35°C and 85.58 ± 0.23°C, respectively, using LCGreen^® PLUS.     

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.     

DNA Analysis of Natural Fiber Rope*

Abstract: When rope is found at a crime scene, the type of fiber is currently identified through its microscopic characteristics. However, these characteristics may not always unambiguously distinguish some types of rope from others. If rope samples contain cells from the plants of origin, then DNA analysis may prove to be a better way to identify the type of rope obtained from a crime scene. The objective of this project was to develop techniques of DNA analysis that can be used to differentiate between ropes made from Cannabis sativa L. (hemp), Agave sisalana Perrine (sisal), Musa textilis Née (abaca, “Manila hemp”), Linum usitatissimum L. (flax), and Corchorus olitorus L. (jute). The procedures included extracting the DNA from the rope, performing polymerase chain reaction (PCR) using the extracted DNA as a template, and analyzing the DNA products. A primer pair for PCR, chosen from within a chloroplast gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase, was designed to be specific for plant DNA and complementary to the genes from all five plants. The resulting PCR fragments were approximately 771 base pairs long. The PCR fragments, distinguished through base sequence analysis or restriction enzyme analysis, could be used to identify the five different rope types. The procedure provides a useful addition to visual methods of comparing rope samples.     

Are these food products fraudulent? Rapid and novel triplex-direct PCR assay for meat identification

Unintentional contamination and fraudulent labeling of food, especially in halal-certified products, breach both religious and international laws. DNA-based methods are widely employed to identify trace amount of contaminants for law enforcement. Direct PCR has proved successful in the DNA analysis from degraded samples and PCR-inhibited samples, but it has never been applied to meat identification from food products. In this study, we aimed to develop a multiplex direct PCR assay for simultaneous identification of three commonly consumed meats without the need to extract DNA. Species-specific primers were designed from the mitochondrial DNA using the alignment of sequences available on GenBank. The assay was validated for its specificity, sensitivity, and usefulness in market sample analysis. The results showed that a highly specific and sensitive multiplex direct PCR assay was developed and provided the expected PCR fragment of approximately 100, 119, and 133 bp for pork (Sus scrofa), mutton (Ovis aries), and chicken (Gallus gallus), respectively. Thirty-nine market samples were tested and a small number of fraudulent labeling was detected. In conclusion, we developed a rapid and inexpensive test for three meat species. This cost- and time-saving assay could be easily adopted in the world food hubs, which are mostly third-world countries.     

Forensic Identification of CITES Protected Slimming Cactus (Hoodia) Using DNA Barcoding

Slimming cactus (Hoodia), found only in southwestern Africa, is a well‐known herbal product for losing weight. Consequently, Hoodia extracts are sought‐after worldwide despite a CITES Appendix II status. The failure to eradicate illegal trade is due to problems with detecting and identifying Hoodia using morphological and chemical characters. Our aim was to evaluate the potential of molecular identification of Hoodia based on DNA barcoding. Screening of nrITS1 and psbA‐trnH DNA sequences from 26 accessions of Ceropegieae resulted in successful identification, while conventional chemical profiling using DLI‐MS led to inaccurate detection and identification of Hoodia. The presence of Hoodia in herbal products was also successfully established using DNA sequences. A validation procedure of our DNA barcoding protocol demonstrated its robustness to changes in PCR conditions. We conclude that DNA barcoding is an effective tool for Hoodia detection and identification which can contribute to preventing illegal trade.     

Molecular Identification of Necrophagous Dermestes Species in South Korea Using Cytochrome c Oxidase Subunit I Nucleotide Sequences (Genus Dermestes)

Species identification of necrophagous insects found on a dead body is an essential key in applying medicolegal entomology to the estimation of postmortem interval (PMI). Due to limited morphological identification of insect evidence, several studies have identified species using molecular information such as DNA markers. While considerable cytochrome c oxidase subunit I (COI) gene sequence data of necrophagous fly species have been collected and annotated, those of necrophagous beetle species have not. Since necrophagous beetles such as Dermestes species have a larval period longer than that of flies, beetles are useful in even the late decomposition phase in estimating minimum PMI. To obtain the full-length COI gene sequences of six Dermestes species collected from South Korea, we designed primers for polymerase chain reaction amplification and sequencing. The obtained full COI nucleotide sequences were used for performing phylogenic analysis and comparison with previously reported sequences. The results demonstrated that the COI gene sequences could be used to identify forensically important Dermestes species in South Korea.     

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.     

Identification of mammal species using species-specific DNA pyrosequencing

In forensic casework it is highly relevant to be able to deduce the species origin of an unknown biological sample. For such a purpose we have designed and developed an assay for species identification based on DNA sequencing of two short mitochondrial DNA amplicons. In short, partial 12S rRNA and partial 16S rRNA fragments (approximately 100bp) are amplified by PCR followed by direct sequencing using pyrosequencing technique. Due to properties of the chosen targets, the same PCR conditions and primers were used irrespective of the true species of an unknown sample. A total of 28 different mammals present in the European fauna were sequenced both for the partial 12S rRNA and the partial 16S rRNA sequences for accuracy verification. Together the two sequences showed to have a high divergence factor, discriminating almost all mammals. Furthermore, the human reference nucleotide sequences were always at least nine nucleotides different compared to the other sequenced species both at the partial 12S rRNA and the partial 16S rRNA sequences.     

Organelle DNA haplotypes reflect crop-use characteristics and geographic origins of Cannabis sativa

Comparative sequencing of cannabis individuals across 12 chloroplast and mitochondrial DNA loci revealed 7 polymorphic sites, including 5 length variable regions and 2 single nucleotide polymorphisms. Simple PCR assays were developed to assay these polymorphisms, and organelle DNA haplotypes were obtained for 188 cannabis individuals from 76 separate populations, including drug-type, fibre-type and wild populations. The haplotype data were analysed using parsimony, UPGMA and neighbour joining methods. Three haplotype groups were recovered by each analysis method, and these groups are suggestive of the crop-use characteristics and geographical origin of the populations, although not strictly diagnostic. We discuss the relationship between our haplotype data and taxonomic opinions of cannabis, and the implications of organelle DNA haplotyping to forensic investigations of cannabis.     

改良高盐低pH方法提取陈旧大麻DNA初探

目的探讨建立室温保存10年队上大麻干叶及大麻树脂DNA提取方法。方法采用SDS及改良高盐低pH方法，改变提取缓冲液中β-巯基乙醇终浓度，增加用酚、氯仿快速抽提过程，提取新鲜和陈旧大麻（树脂）DNA，应用大麻叶绿体trnLintron引物进行PCR，琼脂糖凝胶电泳法检测扩增产物。结果用高盐低pH方法获得了10年以上大麻干叶及树脂清晰的电泳图谱，其中成功提取了1份23年陈旧大麻的DNA。结论高盐低pH方法操作简便、实用，可望用于陈旧、微量大麻植株的DNA检测，对于涉毒案件中特殊大麻标本的检验具有一定意义。