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。     

基于rbcL序列的大麻鉴定

目的评估叶绿体DNA rbc L序列作为遗传标记鉴定大麻的可行性。方法检测62份大麻、10份啤酒花及10份葎草DNA的rbc L序列,并从Gen Bank数据库中下载96条大麻科rbc L序列。应用MEGA X软件进行序列比对,计算种内及种间Kimura-2-Parameter(K2P)遗传距离并构建系统聚类树。结果本次大麻及葎草属样本测序所得rbc L序列长度分别为617 bp和649 bp,且在所测大麻样本中检测到2种单倍型。BLAST相似性检索结果显示,测序所得序列与Gen Bank数据库中大麻rbc L序列相似性最高为100%。遗传距离分析结果显示,大麻种内不同样本间的最大遗传距离(0.004 9)小于大麻与大麻科其他物种间的最小遗传距离(0.012 9)。从中介网络图和系统聚类分析中可以看出,大麻与大麻科其他物种位于不同分支。结论 rbc L序列可以作为鉴定大麻的DNA条形码,联合rbc L序列的比对分析和系统聚类分析有望成为法医学大麻种属鉴定可靠、便捷的检测手段。     

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。     

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。     

云南汉族人群19个STR基因座遗传多态性

本文对中国云南地区汉族2 384个健康无关个体进行19个STR基因座遗传多态性调查。采用Chelex-100法提取样本DNA,用Goldeneye TM 20A试剂盒进行扩增及检测。结果在19个STR基因座共检出266个等位基因和1246种基因型,其分布符合Hardy-Weinberg平衡定律(P0.05)。所调查的云南汉族人群19个STR基因座具有较好识别能力。     

云南苗族群体15个STR基因座遗传多态性

本文对中国云南地区苗族219个健康无关个体进行15个常染色体STR基因座遗传多态性调查。采用Chelex-100法提取样本DNA,用Power Plex16 System试剂盒进行扩增及检测。结果在15个STR基因座共检出161个等位基因和438种基因型,其分布符合Hardy-Weinberg平衡定律(P0.05)。所调查的云南人群15个STR基因座具有较好识别能力。     

云南德宏缅籍人群15个常染色体STR基因座遗传多态性

本文对中国云南德宏缅族人群1 072个健康无关个体进行15个STR基因座遗传多态性调查。采用Chelex-100法提取样本DNA,用Amp F.STR Sinofiler试剂盒进行扩增及检测。结果在15个STR基因座共检出195个等位基因和813种基因型,其分布符合Hardy-Weinberg平衡定律(P0.05)。所调查的云南德宏缅族人群15个STR基因座具有较好识别能力。     

利用DNAITS2条形码序列鉴定植物大麻和罂粟

目的分析毒品原植物大麻、罂粟及其混伪品植物r DNA的ITS2序列信息,探索鉴定大麻、罂粟及其混伪品的新方法。方法采用PCR法扩增ITS2序列,双向测序后运用Codon Code Aligner、MEGA5.1软件进行数据处理,计算种内种间K2P距离,构建系统聚类树(NJ树)。结果大麻、罂粟的种内K2P遗传距离为0,大麻、罂粟及其混伪品之间的最小K2P距离为0.187。NJ树表现出明显的单系性。结论利用DNA ITS2条形码序列有可能鉴定出毒品原植物大麻、罂粟。     

MiSeq FGx~(TM)系统进行基因突变亲权鉴定1例

<正>1材料与方法1.1 DNA提取及定量样本来源于本实验室日常案件中经ID-PLUS检测存在STR遗传位点突变的三联体的FTA唾液口腔卡,并使用Qiagen DNA Investigator试剂盒(Qiagen公司)提取样本DNA。样本DNA采用Qubit?3.0荧光定量仪(Life Technologies公司)进行DNA浓度检测,并将浓度稀释到0.2ng/μL[1]。     

云南地区彝族人群17个STR基因座的遗传多态性

本文对中国云南地区彝族390个健康无关个体进行17个STR基因座遗传多态性调查。采用Chelex-100法提取样本DNA,用AGCU 17+1 STR荧光检测试剂盒进行扩增及检测。上述17个STR基因座共检出191个等位基因和704种基因型,其分布符合Hardy-Weinberg平衡定律(P0.05),在所调查的彝族人群具有较好识别能力。     

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.     

Cannabis Profiling Based on Its Elemental Composition—Is It Possible?

Abstract: Elemental composition of 85 cannabis samples was established using GF AAS and ICP OES methods. The robustness of the method was determined by analyzing eight independently prepared replicates from a single cannabis plant. The accuracy of the method was established by analyzing four plant certified reference material samples. The ability of discriminant analysis using elemental compositions to distinguish between fiber cannabis samples collected from four different regions of Poland was evaluated. Then, a classification model was developed that correctly classified selected samples of known origin. Cannabis samples confiscated by law enforcement agencies have also been subjected to discriminant analysis. A classification model has been developed for four locations in Poland (Bia?ystok, Ko?cierzyna, the environs of Skar?ysko Kamienna, and Bydgoszcz), to help determine where samples of unknown origin could have been grown.     

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.     

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.     

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.     

Potency levels of regulated cannabis products in Michigan 2021–2022

Evaluation of cannabinoid concentrations in products from the legal cannabis market has been fraught with uncertainty. The lack of standardized testing methodology and the susceptibility of cannabinoids to degradation under certain storage conditions complicates the efforts to assess total tetrahydrocannabinol (THC) levels across wide geographic areas. There are few peer-reviewed surveys of cannabinoid concentrations in regulated products. Those that have been done have not characterized the effects of differences in analytical methodology, sample population, and storage conditions. Viridis Laboratories, which operates two cannabis safety compliance facilities in Michigan, has analyzed over 34,000 cannabis products throughout 2021 and 2022 before the sale in the regulated market. Fifteen cannabinoids in cannabis flower, concentrates, and infused products were tested using methanolic extraction and analysis by high-performance liquid chromatography with diode-array detection. Methods were validated before use, and the flower analysis procedure was certified by the Association of Analytical Collaboration. All the samples were tested before submission for sale and therefore had not undergone prolonged storage. The results are compared with those seen in other states as well as in the illicit market. Total THC levels in cannabis flower from the regulated market are significantly higher than those seen in illicit products. The distribution of cannabinoid levels is similar in flowers intended for either the medicinal or adult-use markets, with an average potency of 18%–23% of total THC. Total THC in concentrates averages up to 82%. Other cannabinoids are observed at significant levels, mostly in products specifically formulated to contain them. These results may act as a benchmark for potency levels in the regulated market.     

The potency of cannabis in New Zealand from 1976 to 1996.

The potency of cannabis plant and cannabis products seized in New Zealand over the period of 20 years is studied. The earlier part of the study includes mainly imported cannabis oil and cannabis resin, and both imported and locally grown cannabis plant, that was seized by the police. The later part of the study includes little imported material. Cannabis plant is now locally grown, cannabis oil is locally manufactured and imported cannabis resin is rarely seized. The average potency of the cannabis plant available to the user has not increased over the 20 years period. Cannabis leaf contains on average 1% THC and the female flowering heads on average 3.5% THC. The average potency of cannabis oil has dropped from its peak at 34% THC in 1985 to 13% THC in 1995.     

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.     

Short tandem repeat (STR) DNA markers are hypervariable and informative in Cannabis sativa: implications for forensic investigations

Short tandem repeat (STR) markers are the DNA marker of choice in forensic analysis of human DNA. Here we extend the application of STR markers to Cannabis sativa and demonstrate their potential for forensic investigations. Ninety-three individual cannabis plants, representing drug and fibre accessions of widespread origin were profiled with five STR makers. A total of 79 alleles were detected across the five loci. All but four individuals from a single drug-type accession had a unique multilocus genotype. An analysis of molecular variance (AMOVA) revealed significant genetic variation among accessions, with an average of 25% genetic differentiation. By contrast, only 6% genetic difference was detected between drug and fibre crop accessions and it was not possible to unequivocally assign plants as either drug or fibre type. However, our results suggest that drug strains may typically possess lower genetic diversity than fibre strains, which may ultimately provide a means of genetic delineation. Our findings demonstrate the promise of cannabis STR markers to provide information on: (1) agronomic type, (2) the geographical origin of drug seizures, and (3) evidence of conspiracy in production of clonally propagated drug crops.