本文選題：大麻 + STR　； 參考：《河北醫(yī)科大學(xué)》2007年博士論文

【摘要】： 大麻是一種古老的栽培植物,是許多國(guó)家重要的經(jīng)濟(jì)作物。但因其含有毒性成分—四氫大麻酚(THC)和大麻二醇(CBD),已被聯(lián)合國(guó)禁毒公約列為與海洛因、可卡因并列的三大毒品之一。近年來(lái),毒品問(wèn)題日益嚴(yán)重。無(wú)論是在中國(guó),還是在世界范圍內(nèi),其都對(duì)人類的生存和社會(huì)發(fā)展構(gòu)成了嚴(yán)重的威脅。因此,禁毒是擺在世界人民面前的一項(xiàng)迫切任務(wù),而這又是一項(xiàng)極其復(fù)雜的系統(tǒng)工程,需要多部門、多領(lǐng)域、多學(xué)科的相互配合。這其中非常重要的一種手段就是應(yīng)用現(xiàn)代的科學(xué)檢驗(yàn)技術(shù)對(duì)繳獲的毒品進(jìn)行定性和定量,以便確定其來(lái)源,從而使根除毒源成為可能。目前主要是通過(guò)傳統(tǒng)的化學(xué)方法和生物學(xué)手段來(lái)分析大麻中的毒性成份從而對(duì)其進(jìn)行定性和定量并推斷毒源。這些方法在檢測(cè)中常常需要大量的大麻樣品,而且必須是新鮮的,因?yàn)樗臍浯舐榉?THC)在陳舊標(biāo)本中很容易被氧化,使檢驗(yàn)具有一定的局限性。 在大麻毒品的案件中,常見的檢材是不同條件下放置的干葉或發(fā)霉、腐敗的陳舊標(biāo)本,還有一些含脂類物質(zhì)較多的大麻樹脂產(chǎn)品。這些檢材在獲得時(shí)多半是被曬干、切碎的大麻制品,肉眼觀察很難與茶葉等外觀相近的植物進(jìn)行分辨。在大麻犯罪案件中還經(jīng)常繳獲到成批未加工的大麻植物,可能來(lái)自于同一產(chǎn)地的大麻品種,或來(lái)自于不同產(chǎn)地的不同大麻品種,如能鑒別出這些大麻的品種,則能推斷所繳大麻的毒源地,為鏟除毒品源頭提供重要幫助。 本研究旨在針對(duì)法庭科學(xué)中常見的毒品原植物—大麻標(biāo)本的特點(diǎn),初步建立適用于實(shí)際辦案的快速、準(zhǔn)確、經(jīng)濟(jì)、方便的大麻植物基因組DNA的提取及擴(kuò)增檢測(cè)方法,通過(guò)DNA分析技術(shù)檢測(cè)大麻的遺傳多態(tài)性,并能有效的鑒別、鑒定毒品原植物—大麻。目前利用DNA分析技術(shù)來(lái)檢測(cè)毒品原植物的特性并鑒定不同產(chǎn)地的毒品原植物正逐漸成為禁毒工作中一項(xiàng)重要內(nèi)容,為推斷毒品的來(lái)源開辟了一條新的技術(shù)途徑。這個(gè)新技術(shù)的開展將會(huì)推動(dòng)法庭科學(xué)對(duì)毒物毒品的深入研究,對(duì)于禁止毒品流行、禁種禁吸以及對(duì)于基層辦案和對(duì)毒品的檢驗(yàn)工作都具有一定的實(shí)際意義和使用價(jià)值。 本實(shí)驗(yàn)所開展的工作是國(guó)家十一五支撐計(jì)劃項(xiàng)目的前期內(nèi)容,其為后續(xù)的深入研究奠定了基礎(chǔ),填補(bǔ)了國(guó)內(nèi)毒品原植物DNA檢驗(yàn)在法庭科學(xué)應(yīng)用的空白。 1新鮮大麻基因組DNA提取方法的建立及大麻特異遺傳片段的篩選 目的:用改良的SDS方法對(duì)新鮮大麻嫩葉進(jìn)行基因組DNA提取及檢測(cè)的方法,并篩選出對(duì)大麻特異的遺傳片段,建立一種穩(wěn)定、靈敏、實(shí)用的毒品原植物大麻DNA檢驗(yàn)的方法,為大麻涉毒案件的來(lái)源推斷奠定基礎(chǔ)。 方法:對(duì)傳統(tǒng)的SDS方法略有改動(dòng),通過(guò)改變提取緩沖液中β-巰基乙醇的終濃度提取了5個(gè)品種共20份(雌、雄)新鮮大麻標(biāo)本的基因組DNA。在提取過(guò)程中,先將標(biāo)本用液氮速凍后粉碎,再經(jīng)研磨釋放DNA然后用SDS裂解緩沖液抽提。提取到的大麻基因組DNA用核酸蛋白定量?jī)x進(jìn)行定量,用所篩選的引物進(jìn)行PCR擴(kuò)增,經(jīng)瓊脂糖凝膠電泳進(jìn)行檢測(cè)大麻的基因組DNA。同時(shí)對(duì)大麻特異性擴(kuò)增產(chǎn)物進(jìn)行了測(cè)序。 結(jié)果:使用改良SDS方法提取新鮮大麻植物標(biāo)本DNA,獲得了清晰的凝膠電泳圖譜,從候選的5對(duì)植物通用引物中篩選出一對(duì)大麻的特異引物,經(jīng)測(cè)序后確定為約190bp大小的片段。應(yīng)用該對(duì)引物擴(kuò)增非大麻類植物參照標(biāo)本,未檢測(cè)到擴(kuò)增產(chǎn)物。 結(jié)論:選用改良SDS法提取新鮮大麻植物標(biāo)本DNA,可以獲得高質(zhì)量的大麻基因組DNA,足以用于進(jìn)一步檢測(cè)所需。用所篩選的大麻特異遺傳片段可以鑒別大麻與其它植物的種屬,對(duì)毒品案件的鑒定提供了一種實(shí)用、有效的方法。 2特殊大麻標(biāo)本DNA提取方法的建立及不同方法的比較 目的:初步建立室溫保存10年以上大麻干葉子及大麻樹脂的基因組DNA提取方法。建立穩(wěn)定的從降解嚴(yán)重陳舊的大麻標(biāo)本獲得高質(zhì)量DNA的提取方法。 方法:用SDS法、CTAB法、改良高鹽低pH法、堿裂解法及磁珠純化的方法對(duì)特殊陳舊大麻檢材進(jìn)行基因組DNA提取,通過(guò)比較以確定最佳的提取方法。即高鹽低pH方法,提取了新鮮大麻標(biāo)本和陳舊大麻(樹脂)標(biāo)本的DNA,模板DNA經(jīng)定量后選擇最適濃度為5～10ng/μl,應(yīng)用大麻葉綠體trnL intron的引物進(jìn)行PCR擴(kuò)增,經(jīng)瓊脂糖凝膠電泳方法檢測(cè)擴(kuò)增產(chǎn)物。 結(jié)果:在SDS法、CTAB法、改良高鹽低pH法、堿裂解法這4種提取大麻基因組DNA方法中,尤以改良高鹽低pH方法最佳,得到了純度較高、片段較完整的大麻基因組DNA。經(jīng)檢測(cè)獲得了10年以上大麻干葉及大麻樹脂的清晰凝膠電泳圖譜,其中成功提取了1份22年陳舊大麻樹脂標(biāo)本的DNA。 結(jié)論:該方法操作簡(jiǎn)便、實(shí)用,對(duì)于陳舊、降解的特殊大麻標(biāo)本及大麻樹脂產(chǎn)品DNA的提取效果尤佳。同時(shí)該方法可用于檢測(cè)大麻植株來(lái)源,對(duì)于法庭科學(xué)的研究和實(shí)際檢案均具有重要的意義。 3特異的大麻性別位點(diǎn)研究 目的:設(shè)計(jì)對(duì)大麻雄性植株特異的引物,以鑒別具有經(jīng)濟(jì)價(jià)值的大麻雄性植株,尤其在大麻幼苗期能夠區(qū)分開具有藥用價(jià)值和濫用潛力的大麻雌性植株和經(jīng)濟(jì)價(jià)值的雄性植株,以期在毒品案件中能從DNA水平上識(shí)別大麻雌、雄樣本。 方法:用SDS法、改良高鹽低pH法和磁珠法提取新鮮和陳舊大麻雌、雄標(biāo)本及大麻樹脂標(biāo)本的基因組DNA,使用“Primer Premier 5.0”軟件設(shè)計(jì)引物PCR擴(kuò)增,經(jīng)瓊脂糖凝膠電泳檢測(cè)擴(kuò)增產(chǎn)物。 結(jié)果:自行設(shè)計(jì)的引物經(jīng)PCR擴(kuò)增得到約300~320bp大小的產(chǎn)物片斷,在電泳檢測(cè)時(shí),只有Ym21、Ym23、Ym26、Ym71四種雄性大麻標(biāo)本全部出現(xiàn)了清晰的譜帶,而同樣條件下擴(kuò)增的六種陳舊雌性大麻標(biāo)本均未檢測(cè)到譜帶。 結(jié)論:初步認(rèn)為這對(duì)引物的擴(kuò)增片斷長(zhǎng)度在大麻植株雌、雄性別間存在差異。但尚需分析更多的標(biāo)本,并進(jìn)行序列測(cè)定,進(jìn)一步明確引物的種屬特異性及性別特異性。 4大麻STR位點(diǎn)的遺傳多態(tài)性研究 目的:研究CS1和ANUCS305 STR位點(diǎn)在中國(guó)不同地區(qū)大麻中的遺傳多態(tài)性,并探討其在法庭科學(xué)中的應(yīng)用。建立穩(wěn)定、靈敏、實(shí)用的大麻STR位點(diǎn)的分析檢測(cè)方法,對(duì)不同大麻品種的個(gè)體進(jìn)行遺傳多樣性分析,檢驗(yàn)結(jié)果能夠有效的鑒別、鑒定毒品原植物大麻。通過(guò)研究初步嘗試?yán)肧TR分析技術(shù)推斷涉毒案件中所繳大麻的來(lái)源地。 方法:以5-FAM熒光染料標(biāo)記設(shè)計(jì)、改進(jìn)、篩選的CS1和ANUCS305兩個(gè)STR位點(diǎn),對(duì)云南大麻四個(gè)品種62株個(gè)體進(jìn)行PCR擴(kuò)增,通過(guò)ABI310/3100毛細(xì)管電泳基因型分析儀,建立快速、高通量的熒光檢測(cè)體系,使用GenAlEx6軟件進(jìn)行遺傳多態(tài)性分析。 結(jié)果:兩個(gè)STR位點(diǎn)的多態(tài)性均較高,其等位基因數(shù)在7-26之間,多態(tài)信息含量在0.43-0.90之間,有效等位基因數(shù)在2.040-10.449之間,雜合度在0.250-0.923之間。通過(guò)GenAlEx6軟件分別計(jì)算出了四個(gè)云麻品種之間的Nei's遺傳距離,然后使用UPGMA法繪制了云麻四個(gè)品種組成的系統(tǒng)聚類圖。 結(jié)論:所選CS1和ANUCS305STR位點(diǎn)具有較高的遺傳多態(tài)性,若能開發(fā)出更多此類位點(diǎn),并能采集較多不同來(lái)源地的標(biāo)本進(jìn)行分析建檔,以此建立起完整的全國(guó)大麻稀有等位基因數(shù)據(jù)庫(kù)和各品種間聚類系統(tǒng)樹,可使大麻品種間的鑒定成為可能,同時(shí)也能提高推斷毒品原植物大麻的來(lái)源地及其在實(shí)際案件的刑事鑒定上的應(yīng)用價(jià)值。 結(jié)論 本研究從新鮮大麻DNA提取及特異引物的篩選、特殊大麻標(biāo)本DNA提取、特異的大麻性別位點(diǎn)、大麻STR位點(diǎn)的遺傳多態(tài)性等幾個(gè)方面對(duì)毒品原植物大麻的DNA檢測(cè)技術(shù)進(jìn)行了的研究,建立了大麻DNA提取及檢測(cè)方法,對(duì)涉毒案件中毒品原植物的檢驗(yàn)奠定了基礎(chǔ)。 1本研究選用SDS法提取新鮮大麻植物標(biāo)本DNA,可以獲得高質(zhì)量的大麻基因組DNA。 2本研究對(duì)所選擇的幾種提取特殊大麻標(biāo)本的方法進(jìn)行了研究,實(shí)驗(yàn)表明改良高鹽低pH方法最佳,得到了純度較高、片段較完整的大麻基因組DNA。并獲得了10年以上大麻干葉及大麻樹脂的清晰凝膠電泳圖譜。 3本研究自行設(shè)計(jì)的一對(duì)大麻雄性引物,經(jīng)PCR擴(kuò)增得到300~350bp大小的產(chǎn)物片斷,電泳檢出雄性大麻標(biāo)本的清晰譜帶,而未檢測(cè)到同樣條件擴(kuò)增的雌性大麻標(biāo)本和非大麻植物對(duì)照標(biāo)本的譜帶。 4本研究對(duì)毒品原植物大麻的CS1和ANUCS305STR兩個(gè)STR位點(diǎn)多態(tài)性進(jìn)行了研究,并通過(guò)GenAlEx6軟件分別計(jì)算出了四個(gè)云南大麻品種之間的Nei's遺傳距離,然后使用UPGMA法繪制了云南大麻四個(gè)品種組成的系統(tǒng)聚類圖。研究結(jié)果證實(shí):這兩個(gè)STR位點(diǎn)具有較高的遺傳多態(tài)性,其等位基因數(shù)在7-26之間,多態(tài)信息含量在0.43-0.90之間,有效等位基因數(shù)在2.040-10.449之間,雜合度在0.250-0.923之間。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：D919

【引證文獻(xiàn)】

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1 張多勇;李云;;從考古發(fā)現(xiàn)看馬家窯人的生產(chǎn)活動(dòng)[J];農(nóng)業(yè)考古;2012年06期

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1 苗翠英;;毒品檢測(cè)新技術(shù)[A];公共安全中的化學(xué)問(wèn)題研究進(jìn)展（第二卷）[C];2011年

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本文編號(hào)：1989678