毒品原植物大麻DNA检测技术的研究

发布时间：2018-06-07 04:03

本文选题：大麻 + STR　；参考：《河北医科大学》2007年博士论文

【摘要】： 大麻是一种古老的栽培植物,是许多国家重要的经济作物。但因其含有毒性成分—四氢大麻酚(THC)和大麻二醇(CBD),已被联合国禁毒公约列为与海洛因、可卡因并列的三大毒品之一。近年来,毒品问题日益严重。无论是在中国,还是在世界范围内,其都对人类的生存和社会发展构成了严重的威胁。因此,禁毒是摆在世界人民面前的一项迫切任务,而这又是一项极其复杂的系统工程,需要多部门、多领域、多学科的相互配合。这其中非常重要的一种手段就是应用现代的科学检验技术对缴获的毒品进行定性和定量,以便确定其来源,从而使根除毒源成为可能。目前主要是通过传统的化学方法和生物学手段来分析大麻中的毒性成份从而对其进行定性和定量并推断毒源。这些方法在检测中常常需要大量的大麻样品,而且必须是新鲜的,因为四氢大麻酚(THC)在陈旧标本中很容易被氧化,使检验具有一定的局限性。在大麻毒品的案件中,常见的检材是不同条件下放置的干叶或发霉、腐败的陈旧标本,还有一些含脂类物质较多的大麻树脂产品。这些检材在获得时多半是被晒干、切碎的大麻制品,肉眼观察很难与茶叶等外观相近的植物进行分辨。在大麻犯罪案件中还经常缴获到成批未加工的大麻植物,可能来自于同一产地的大麻品种,或来自于不同产地的不同大麻品种,如能鉴别出这些大麻的品种,则能推断所缴大麻的毒源地,为铲除毒品源头提供重要帮助。本研究旨在针对法庭科学中常见的毒品原植物—大麻标本的特点,初步建立适用于实际办案的快速、准确、经济、方便的大麻植物基因组DNA的提取及扩增检测方法,通过DNA分析技术检测大麻的遗传多态性,并能有效的鉴别、鉴定毒品原植物—大麻。目前利用DNA分析技术来检测毒品原植物的特性并鉴定不同产地的毒品原植物正逐渐成为禁毒工作中一项重要内容,为推断毒品的来源开辟了一条新的技术途径。这个新技术的开展将会推动法庭科学对毒物毒品的深入研究,对于禁止毒品流行、禁种禁吸以及对于基层办案和对毒品的检验工作都具有一定的实际意义和使用价值。本实验所开展的工作是国家十一五支撑计划项目的前期内容,其为后续的深入研究奠定了基础,填补了国内毒品原植物DNA检验在法庭科学应用的空白。 1新鲜大麻基因组DNA提取方法的建立及大麻特异遗传片段的筛选目的:用改良的SDS方法对新鲜大麻嫩叶进行基因组DNA提取及检测的方法,并筛选出对大麻特异的遗传片段,建立一种稳定、灵敏、实用的毒品原植物大麻DNA检验的方法,为大麻涉毒案件的来源推断奠定基础。方法:对传统的SDS方法略有改动,通过改变提取缓冲液中β-巯基乙醇的终浓度提取了5个品种共20份(雌、雄)新鲜大麻标本的基因组DNA。在提取过程中,先将标本用液氮速冻后粉碎,再经研磨释放DNA然后用SDS裂解缓冲液抽提。提取到的大麻基因组DNA用核酸蛋白定量仪进行定量,用所筛选的引物进行PCR扩增,经琼脂糖凝胶电泳进行检测大麻的基因组DNA。同时对大麻特异性扩增产物进行了测序。结果:使用改良SDS方法提取新鲜大麻植物标本DNA,获得了清晰的凝胶电泳图谱,从候选的5对植物通用引物中筛选出一对大麻的特异引物,经测序后确定为约190bp大小的片段。应用该对引物扩增非大麻类植物参照标本,未检测到扩增产物。结论:选用改良SDS法提取新鲜大麻植物标本DNA,可以获得高质量的大麻基因组DNA,足以用于进一步检测所需。用所筛选的大麻特异遗传片段可以鉴别大麻与其它植物的种属,对毒品案件的鉴定提供了一种实用、有效的方法。 2特殊大麻标本DNA提取方法的建立及不同方法的比较目的:初步建立室温保存10年以上大麻干叶子及大麻树脂的基因组DNA提取方法。建立稳定的从降解严重陈旧的大麻标本获得高质量DNA的提取方法。方法:用SDS法、CTAB法、改良高盐低pH法、碱裂解法及磁珠纯化的方法对特殊陈旧大麻检材进行基因组DNA提取,通过比较以确定最佳的提取方法。即高盐低pH方法,提取了新鲜大麻标本和陈旧大麻(树脂)标本的DNA,模板DNA经定量后选择最适浓度为5～10ng/μl,应用大麻叶绿体trnL intron的引物进行PCR扩增,经琼脂糖凝胶电泳方法检测扩增产物。结果:在SDS法、CTAB法、改良高盐低pH法、碱裂解法这4种提取大麻基因组DNA方法中,尤以改良高盐低pH方法最佳,得到了纯度较高、片段较完整的大麻基因组DNA。经检测获得了10年以上大麻干叶及大麻树脂的清晰凝胶电泳图谱,其中成功提取了1份22年陈旧大麻树脂标本的DNA。结论:该方法操作简便、实用,对于陈旧、降解的特殊大麻标本及大麻树脂产品DNA的提取效果尤佳。同时该方法可用于检测大麻植株来源,对于法庭科学的研究和实际检案均具有重要的意义。 3特异的大麻性别位点研究目的:设计对大麻雄性植株特异的引物,以鉴别具有经济价值的大麻雄性植株,尤其在大麻幼苗期能够区分开具有药用价值和滥用潜力的大麻雌性植株和经济价值的雄性植株,以期在毒品案件中能从DNA水平上识别大麻雌、雄样本。方法:用SDS法、改良高盐低pH法和磁珠法提取新鲜和陈旧大麻雌、雄标本及大麻树脂标本的基因组DNA,使用“Primer Premier 5.0”软件设计引物PCR扩增,经琼脂糖凝胶电泳检测扩增产物。结果:自行设计的引物经PCR扩增得到约300~320bp大小的产物片断,在电泳检测时,只有Ym21、Ym23、Ym26、Ym71四种雄性大麻标本全部出现了清晰的谱带,而同样条件下扩增的六种陈旧雌性大麻标本均未检测到谱带。结论:初步认为这对引物的扩增片断长度在大麻植株雌、雄性别间存在差异。但尚需分析更多的标本,并进行序列测定,进一步明确引物的种属特异性及性别特异性。 4大麻STR位点的遗传多态性研究目的:研究CS1和ANUCS305 STR位点在中国不同地区大麻中的遗传多态性,并探讨其在法庭科学中的应用。建立稳定、灵敏、实用的大麻STR位点的分析检测方法,对不同大麻品种的个体进行遗传多样性分析,检验结果能够有效的鉴别、鉴定毒品原植物大麻。通过研究初步尝试利用STR分析技术推断涉毒案件中所缴大麻的来源地。方法:以5-FAM荧光染料标记设计、改进、筛选的CS1和ANUCS305两个STR位点,对云南大麻四个品种62株个体进行PCR扩增,通过ABI310/3100毛细管电泳基因型分析仪,建立快速、高通量的荧光检测体系,使用GenAlEx6软件进行遗传多态性分析。结果:两个STR位点的多态性均较高,其等位基因数在7-26之间,多态信息含量在0.43-0.90之间,有效等位基因数在2.040-10.449之间,杂合度在0.250-0.923之间。通过GenAlEx6软件分别计算出了四个云麻品种之间的Nei's遗传距离,然后使用UPGMA法绘制了云麻四个品种组成的系统聚类图。结论:所选CS1和ANUCS305STR位点具有较高的遗传多态性,若能开发出更多此类位点,并能采集较多不同来源地的标本进行分析建档,以此建立起完整的全国大麻稀有等位基因数据库和各品种间聚类系统树,可使大麻品种间的鉴定成为可能,同时也能提高推断毒品原植物大麻的来源地及其在实际案件的刑事鉴定上的应用价值。结论本研究从新鲜大麻DNA提取及特异引物的筛选、特殊大麻标本DNA提取、特异的大麻性别位点、大麻STR位点的遗传多态性等几个方面对毒品原植物大麻的DNA检测技术进行了的研究,建立了大麻DNA提取及检测方法,对涉毒案件中毒品原植物的检验奠定了基础。 1本研究选用SDS法提取新鲜大麻植物标本DNA,可以获得高质量的大麻基因组DNA。 2本研究对所选择的几种提取特殊大麻标本的方法进行了研究,实验表明改良高盐低pH方法最佳,得到了纯度较高、片段较完整的大麻基因组DNA。并获得了10年以上大麻干叶及大麻树脂的清晰凝胶电泳图谱。 3本研究自行设计的一对大麻雄性引物,经PCR扩增得到300~350bp大小的产物片断,电泳检出雄性大麻标本的清晰谱带,而未检测到同样条件扩增的雌性大麻标本和非大麻植物对照标本的谱带。 4本研究对毒品原植物大麻的CS1和ANUCS305STR两个STR位点多态性进行了研究,并通过GenAlEx6软件分别计算出了四个云南大麻品种之间的Nei's遗传距离,然后使用UPGMA法绘制了云南大麻四个品种组成的系统聚类图。研究结果证实:这两个STR位点具有较高的遗传多态性,其等位基因数在7-26之间,多态信息含量在0.43-0.90之间,有效等位基因数在2.040-10.449之间,杂合度在0.250-0.923之间。
[Abstract]:Cannabis, an ancient plant, is an important economic crop in many countries. But because of its toxic ingredients - four THC and CBD, it has been listed as one of the three major drugs with heroin and cocaine by the United Nations Convention on drug control. In recent years, the drug problem has become increasingly serious, both in China and in the world. It is a serious threat to the survival and social development of human beings. Therefore, drug control is an urgent task in front of the people of the world, and this is an extremely complex system engineering which requires multi sector, multi domain and multidisciplinary cooperation. One of the most important means is to apply modern science inspection. It is possible to determine the source of the seized drugs by determining the quality and quantity of the seized drugs, so that it is possible to eradicate the source of poison. It is mainly to analyze the toxic ingredients in cannabis by traditional chemical methods and biological methods to determine and quantify the toxic ingredients and to infer the source of poison. The sample must be fresh, because four hydrogen cannabinoid (THC) is easily oxidized in old samples, making the test have some limitations.
In the case of cannabis, common tests are dried leaves or mouldy, corrupt old specimens, and some cannabis resin products with more lipid substances. These samples are mostly dried, chopped cannabis products, and the naked eye is difficult to distinguish between plants with similar appearance to tea. A lot of unprocessed cannabis plants are often seized in the cases of hemp crimes, which may come from a variety of cannabis varieties in the same origin or from different varieties of cannabis from different habitats. If they can identify the varieties of these cannabis, it can infer the origin of the cannabis and provide important help for the eradication of the source of the drug.
The purpose of this study is to establish a rapid, accurate, economical and convenient method for the extraction and amplification of cannabis plant genome DNA, which is a rapid, accurate, economical and convenient method to detect the genome of cannabis plant, which is a common drug plant in the forensic science, and to detect the genetic polymorphisms of cannabis by DNA analysis, and to identify and identify the original drug plant. At present, the use of DNA technology to detect the characteristics of the drug plants and identify the original plants of different habitats is becoming an important part of the drug control work. It opens up a new technical approach to infer the source of drugs. The development of this new technology will push the forensic science to the deep study of poison and drugs. Prohibition of drug prohibition and prohibition of drug abuse is of practical significance and practical value for grass-roots case handling and drug testing.
The work carried out in this experiment is the preliminary content of the national support plan for 11th Five-Year, which lays the foundation for further further research and fills the gap of the application of DNA test in the domestic drug plant in the court.
1 Extraction of genomic DNA from fresh cannabis and screening of cannabis specific genetic fragments
Objective: to use the modified SDS method to extract and detect the genomic DNA of fresh cannabis leaves, and to screen out the specific genetic fragment of cannabis, and establish a stable, sensitive and practical method for the DNA test of cannabis cannabis, which lays the foundation for the source of cannabis involvement.
Methods: a slight change in the traditional SDS method was made. By changing the final concentration of beta mercapto ethanol in the extraction buffer, the genomic DNA. of 5 varieties of fresh hemp samples (female and male) was extracted, and the sample was extracted with liquid nitrogen in quick freezing, then lapped and released to DNA and then extracted with SDS lysate buffer. Genomic DNA was quantified by a nucleic acid protein quantitative instrument, amplified by PCR, and the genomic DNA. of cannabis was detected by agarose gel electrophoresis and the specific amplification products of cannabis were sequenced.
Results: the modified SDS method was used to extract fresh cannabis plant specimen DNA. A clear gel electrophoresis map was obtained. A pair of specific primers for cannabis were selected from 5 candidate plant general primers. The specific primers were determined to be about 190bp size. The primers were used to amplify the non cannabis reference specimens and the amplified products were not detected.
Conclusion: the high quality cannabis genomic DNA can be obtained by using the modified SDS method to extract the fresh cannabis plant specimen DNA, which can be used for further detection. The specific genetic fragment of the cannabis can be used to identify the species of cannabis and other plants, and the identification of drug cases is provided with a practical and effective method.
2 Establishment of DNA extraction method for special hemp samples and comparison of different methods
Objective: to establish a preliminary method for genomic DNA extraction of cannabis dried leaves and hemp resin preserved at room temperature for more than 10 years. To establish a stable method for obtaining high quality DNA from serious and obsolete hemp specimens.
Methods: SDS, CTAB, modified high salt and low pH, alkaline lysis and magnetic beads were used to extract genomic DNA from special old cannabis samples. By comparison, the best extraction method was determined. That is, high salt and low pH method, the DNA of fresh cannabis specimens and old cannabis (resin) specimens were extracted, and the template DNA was selected to select the optimum concentration after quantitative selection. PCR was amplified by primers of cannabis chloroplast trnL intron from 5 to 10ng/ L, and amplified products were detected by agarose gel electrophoresis.
Results: in SDS, CTAB, improved high salt and low pH method and alkaline lysis, the best method of extracting cannabis genomic DNA was obtained, especially the improved high salt and low pH method. The purity of the genomic DNA. was higher. The clear gel electrophoresis Atlas of hemp dry leaf and large hemp resin was obtained by detecting the complete genome of cannabis genome, which was successfully extracted. 1 specimens of old hemp resin for 22 years DNA.
Conclusion: the method is simple and practical. It is especially good for the extraction of old, degraded special hemp specimens and hemp resin product DNA. This method can be used to detect the source of cannabis plant. It is of great significance to the scientific research and the actual case examination of the tribunal.
Study on 3 specific sex loci of cannabis
Objective: to design the specific primers for cannabis male plants to identify the economic value of the cannabis male plants, especially in the young cannabis seedling stage, which can separate the female plants with medicinal and abuse potential and the male plants of economic value, in order to identify the female and male samples in the drug case from the DNA level.
Methods: the genomic DNA of fresh and old cannabis, male specimens and hemp resin specimens were extracted with SDS method and modified high salt and low pH method and magnetic bead method. The amplified products were amplified by Primer Premier 5 software and amplified by agarose gel electrophoresis.
Results: the self designed primers were amplified by PCR to obtain the fragments of about 300~320bp size. At the time of electrophoresis, only four male hemp specimens of Ym21, Ym23, Ym26, and Ym71 all appeared clear bands, and all six old female hemp specimens under the same conditions were not detected.
Conclusion: it is preliminarily believed that the length of the amplified fragment of this pair of primers is different between the female and the male of the cannabis plant. However, more specimens need to be analyzed and sequenced to further clarify the specificity and sex specificity of the primers.
Study on genetic polymorphism of 4 cannabis STR site
Objective: To study the genetic polymorphism of CS1 and ANUCS305 STR loci in cannabis in different regions of China, and to explore its application in forensic science. To establish a stable, sensitive and practical analysis and detection method for the STR loci of cannabis, and to analyze the genetic diversity of the individuals of different cannabis varieties. The results of the test can be effectively identified and identified. Plant cannabis. Preliminary attempt to infer the source of marijuana in drug-related cases through STR analysis.
Methods: two STR loci of four varieties of cannabis in Yunnan were amplified by 5-FAM fluorescent dye labeling design, improved and screened two STR loci of four cannabis varieties in Yunnan. A rapid and high throughput fluorescence detection system was established by ABI310/3100 capillary electrophoresis genograph analyzer, and genetic polymorphism analysis was carried out by GenAlEx6 software.
Results: the polymorphism of the two STR loci was high, the number of alleles was 7-26, the polymorphism information content was between 0.43-0.90, the number of effective alleles was between 2.040-10.449, and the heterozygosity was between 0.250-0.923. The Nei's genetic distance between four varieties of cloud numbness was calculated by GenAlEx6 software, and then the UPGMA method was used to draw the genetic distance. A systematic cluster diagram of four varieties of hemp.
Conclusion: the selected CS1 and ANUCS305STR loci have high genetic polymorphism. If more such loci can be developed and the samples of many different sources can be collected for analysis, the whole national cannabis rare allele database and cluster system tree can be established, which can make the identification of cannabis varieties possible, At the same time, it can also improve the source of cannabis plant and its application value in criminal identification of actual cases.
conclusion
In this study, DNA extraction of fresh cannabis and specific primers, DNA extraction of special cannabis specimens, specific sex loci of cannabis, genetic polymorphism of cannabis STR locus and other aspects of DNA detection techniques for cannabis cannabis were studied. The method of extraction and detection of cannabis DNA was established. The test laid the foundation.
1 in this study, SDS method was used to extract DNA from fresh cannabis plant specimens, so that high-quality cannabis genome DNA. could be obtained.
2 the methods of extracting special hemp specimens were studied in this study. The experiment showed that the best method was to improve the high salt and low pH method. The high purity and complete genomic DNA. of cannabis were obtained and the clear gel electrophoresis Atlas of hemp dry leaf and hemp resin was obtained for more than 10 years.
3 a pair of cannabis male primers was designed by ourselves. The fragments of 300~350bp size were amplified by PCR. The clear bands of the male hemp specimens were detected by electrophoresis, but the female cannabis specimens and the non cannabis control specimens of the same condition were not detected.
4 the study on the polymorphism of two STR loci of CS1 and ANUCS305STR in drug plant cannabis was studied. The Nei's genetic distance between four Yunnan cannabis varieties was calculated by GenAlEx6 software and UPGMA method was used to map the series of four varieties of cannabis in Yunnan. The results confirmed that these two STR positions were two. The point has high genetic polymorphism, the number of alleles is between 7-26, polymorphism information content is between 0.43-0.90, the number of effective alleles is between 2.040-10.449, and the heterozygosity is between 0.250-0.923.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：D919

【引证文献】