| The hemp is one kind of ancient cultivated plant, and it is an important industrial crop in many countries of the world. Because it includes toxic constituent - tetrahydrocannabinol (THC) and cannabidiol (CBD), it has been juxtaposed with heroin and cocaine one of three main narcotics by the pact of banning drug of United Nations. In recent years the narcotic problem has become more serious, whether in china or in the worldwide scale, narcotics posed frightful threat to humanity's survival and social development. Therefore, banning drugs has become an urgent challenge to the world. It is also an extremely complex system that need the mutual cooperation of many departments. He vital method is the application of modern science and technology to captured narcotics by using qualitative and quantitative analysis, to determine its origin, thus making it possible to eradicate the source of narcotics .The hemp contains tetrahydrocannabinol (THC), an addictive toxic illusion causing constituent. Present analysis of the toxic constituent carried in the hemp on qualitative and the quota is done mainly through the traditional chemistry method. These methods need the massive hemp leaf in the examination, moreover it must be fresh, because tetrahydrocannabinol (THC) oxidize very easily in the older specimens, thus limiting the capability of the experiments.In the hemp narcotics case, usual material is the dry leaf which kept under the dissimilar condition, or the mouldy specimen .The corrupt obsolete specimen, also contains some fat material found in many hemp resin products. These specimens acquired are mostly the hemp by products which are seasoned, cut leaves into pieces, and are very difficult to distinguish from tea and other that have similar outward appearance with hemp. In hemp crime cases specimens are frequently captured in groups of unfinished hemp. These are possibly hemp species from the same habitat, or they may be different species which come from different habitats, If we can distinguish the species of these hemp, then we can infer the source area of the hemp captured, providing great help to eradicate the narcotic source .This research focuses on the characteristics of the common narcotics--Hemp specimens in forensic science, primarily established fast, accurate, economical, and convenient examination methods ,which are suitable for handling a cases for hemp plant DNA extraction and amplification, Through the DNA analysis technology we can examine hemp's genetic polymorpHism, and can effectively identify and distinguish the narcotics original plant. Using the DNA analysis technology to examine the characteristic of narcotic plant and the ability to distinguish the growing locations of the different plants has gradually become a hot spot in the suppression of drug work, opening a new domain that infers the narcotics origin .This new technical development will be able to empower the forensic sciences to develop narcotics research more deeply, and has certain practical significance and value to the narcotic examination work, the prohibition of narcotics prevalent in society, the banning of narcotics cultivation and drug-taking. as well as the basic unit handles a case.1 The establishment of fresh hemp DNA extraction method and hemp special fragment screening.Objective: The research carries on the total DNA extraction and the examination method with the improved SDS method to the tender fresh hemp leaf, and screens special fragments of the hemp. It establishes a stable, sensible, and conventional method of narcotics in original plant hemp DNA examination, and lays the foundation to infer the origin of the hemp in a case .Methods: Slight modification to the traditional SDS method, changing terminal density of beta - SH alcohol in the extraction buffer we extract total DNA from 5 species 20 samples of fresh hemp specimen(female, male). In the extraction process, first a quick-freeze by the liquid nitrogen, then crush and grind, the sample releasing DNA and after the decomposition buffer solution extraction .It withdraws DNA and carries on the quota with the nucleic acid protein quota meter, using the screening primer to carry on PCR, after agarose gel electrophoresis method to examine hemp total DNA, simultaneously uses sequence technique to sequence amplification product of the hemp specific primer.Results: The use of improved SDS method extract DNA from the fresh specimen, has obtained the clear gel electrophoresis atlas, screening a pair of specific primers from 5 pairs of the candidate general primer of plants, after which sequencing determining fragment for approximately 180bp. Using the primer to amplify the non-hemp plant reference specimen, has not found the product.Conclusion: Selecting SDS method to extract DNA from fresh hemp, may obtain high-quality hemp DNA, sufficiently used in further examination. With the special hemp DNA fragment such screens may distinguish the hemp from other kinds of plant, This has provided an effective practical method to the narcotic case.2 The establishment of special hemp specimen DNA extraction method and the comparison of different methodsObjective: The preliminary study and the establishment of DNA extraction method from the leaf and the hemp resin which preserved under the room temperature over 10 years.To seriously degraded hemp specimens it establishes a stable method to obtain quality DNA extraction.Methods: With the SDS method, the CTAB method, uses a high salt low pH method, the alkali decomposition method to extract the total DNA from an obsolete hemp specimen, determines the best extraction method through the comparison. Especially appling improved high salt low pH method, changing terminal density of beta - SH alcohol in the extraction buffer to increase the phenol, the chloroform fast extraction improvement process withdraws DNA from 10 fresh hemp specimens and 8 obsolete hemp (resin) specimens, The application to amplify the primer of hemp chloroplast trnL intron DNA to carry on PCR, after the agarose gel electrophoresis method examination test the product.Results: The results in several inspection procedures, with improved high salt low pH method, obtains the high-purity, complete fragment of total hemp DNA. After the examination obtained clear gel electrophoresis atlas of the hemp leaf and the hemp resin which kept over 10 years, successful extraction of DNA from an obsolete hemp specimen of 23 years.Conclusion: Improved high salt low pH method is simple, practical, and better for the DNA extraction of obsolete, degenerated special hemp specimen and the hemp resin product. Simultaneously this method is available for identifying the origin of hemp, and has the vital significance for actually handling a case regarding forensic science research.3 Special hemp sex locus researchObjective: Design special specific primer to the hemp male adult plant, in order to distinguish female and male adult plants, this can differentiate the hemp female adult plant in the hemp seedling time which has the medicinal value and the potential abuse and the hemp male adult plant which has the industrial crop value. We can expect to distinguish the hemp female and the male sample from the DNA level in the narcotic case .Methods: Using the SDS method and the magnetism bead method we can extract total DNA from fresh hemp female, or male specimen, using the software Primer Premier 5.0 to design one pair of primers, after the agarose gel electrophoresis method to examine the amplification product.Results: Amplify independently designed primer after PCR obtained the 300~350bp fragment, Through electrophoresis examination, only the male hemp specimen appeared in the clear band, but under the similar conditions the female hemp specimens and the non-hemp plant comparison specimens were not detected in the band.Conclusion: Initially we think that there is difference in amplification fragment length of the primer between the adult female hemp and male plants. But we still have to analyze more specimens, and sequence examination will further determine the species specificity and the sex.4 Hemp STR locus genetic polymorphism researchObjective: Studies CS1 and ANUCS305 STR locus in different area of china hemp show genetic polymorphism, and discusses its application in forensic science. It establishes stable, sensitive, and practical analysis examination method of hemp STR locus and analysize the genetic polymorphism of different hemp species. The result can effectively distinguish the narcotics original plant.Methods: Independently designing, improving and screening CS1 and ANUCS305LIANG two STR locus by the 5-FAM fluorescent dye mark, carries on PCR to amplify Yunnan hemp 4 species 62 individuals. Through the ABI310 capillary vessel electrophoresis gene analyzer, we establish fast, high flux fluorescence examination systems, using the GenAlEx6 software to carry on the heredity multiplicity analysis.Results: 2 STR locus have high polymorphism, the number of allele is between 7-26, the content of polymorphism information is between 0.43-0.90, the number of effective allele is between 2.040-10.449, heterozygosity is between 0.250-0.923. Separately calculated Nei's genetic distance between 4 Yunnan hemp species through the GenAlEx6 software. Used the UPGMA method to draw up a system cluster chart composed of Yunnan hemp 4 species.Conclusion: Selected CS1 and ANUCS305 STR locus have high genetic polymorphism. If we can develop more of this kind of locus, and collect many different-origin specimens to analyze and construct files, then we can establish the integral national hemp rare allele database and the cluster genealogical tree of various species. It make it possible to distinguish various hemp species, simultaneously it also can improve the origin inference of narcotics plant ,and its application value in the criminal case.ConclusionThis research study of the narcotic original plant DNA examination technology has several aspects such as the fresh hemp's DNA extraction, the special primer screening, the special hemp specimen DNA extraction, the special hemp sex locus, the hemp STR locus genetic polymorphism. We have established the DNA extraction and examination method, laying the foundation for the original plant's examination in a narcotics case.1 This research selects the improved SDS method to extract DNA from fresh hemp specimen, and obtain high-quality hemp DNA that can be sufficiently used in further examination. Result demonstration: Using this screening, hemp's specific heredity fragment can be distinguished and provides an effective and practical method for the narcotic case.2 During the selected several special hemp specimen extraction methods of this research, the improved high salt low pH method proved the best. It obtains the high-purity, complete fragment of hemp DNA. It also obtains clear gel electrophoresis atlas of the hemp leaf and the hemp resin which kept over 10 years. The results indicated that the operation of improved high salt low pH method is simple, practical and better for the DNA extraction of degenerated hemp specimen and the hemp resin product. This method is available for identifing the origin of hemp, and has vital significance for actually handling a case regarding forensic science research.3 This research independently designs a pair of male hemp primers, amplifying after PCR obtains the 300~350bp fragment product, the agarose gel electrophoresis examines the clear band of the male hemp specimen, but under the similar condition amplifies the female hemp specimen and the non-hemp plant comparison specimen which were not detected in the band. The results indicate that, this pair of primer is specific to hemp's male adult plant.4 This research of CS1 and ANUCS305 STR two STR locus to study the narcotic original plant hemp's polymorphism, Separately calculated Nei's genetic distance between 4 yunnan hemp species through the GenAlEx6 software, and using the UPGMA method to draw up system cluster chart composed of yunnan hemp 4 species. The results confirmed that these 2 STR locus have high polymorphism, The number of allele is between 7-26, the content of polymorphism information is between 0.43-0.90, the number of effective allele is between 2.040-10.449, heterozygosity is between 0.250-0.923. if we can develop much more this kind of locus, and collects many different-origin specimen to analyze and constructs the files, then establishes the integral national hemp rare allele database and the cluster genealogical tree of various species, it make it possible to distinct various hemp species.The original plant narcotic is the source of narcotics manufacturing and trade. This research result proves that using the DNA analysis technology may open a new drug banning domain which is reliable and highly effective. Simultaneously this can act as the important supplement of the narcotics examination method, and it has the extremely vital significance of the prompt discovery and the eradication of narcotics source. |
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