题名 | 大麻STR基因座复合扩增体系的构建及法医学应用研究 |
其他题名 | Development of the STR loci multiplex PCR system of Cannabis sativa and its application in forensic medicine |
作者 | |
学位类型 | 硕士 |
导师 | 李成涛 |
答辩日期 | 2021-06-05 |
学位授予单位 | 温州医科大学 |
学位专业 | 法医学 |
关键词 | 法医遗传学 大麻 STR 复合扩增 毛细管电泳 |
摘要 | 【目的】 1. 构建一个包含 17 个常染色体 STR 基因座和 2 个性别鉴定基因座的大麻 荧光复合扩增检测体系。 2. 评估所构建体系的系统效能和法医学参数,证实其满足司法鉴定中对大 麻种属鉴定、性别鉴定、个体识别及来源地推断的需求。 【方法】 1. 大麻 STR 基因座的筛选。基于文献报道和 GenBank 数据库,查找目前 已开发的大麻 STR 基因座。使用 Primer Premier 5.0 软件和 NCBI 浏览器中 Primer - BLAST 引物设计工具进行引物设计,对设计的引物单点扩增后进行琼脂糖凝胶 电泳,以验证单个基因座引物效果。 2. 大麻 STR 基因座复合扩增荧光检测体系的构建。将筛选到的 17 个常染 色体 STR 基因座和 2 个性别鉴定基因座分成四组,每组各基因座正向引物的 5’ 端分别标记上荧光染料(FAM、HEX、TAMRA 和 ROX)。通过优化各基因座 引物终浓度、复合扩增 PCR 反应体系和反应条件构建五色荧光标记的多重复合 扩增荧光检测体系。根据 3500XL 遗传分析仪的检测需求,建立 5Dye 光谱校正 文件。根据各基因座等位基因的桑格测序结果对其进行命名,并制备等位基因分 型标准品,最后按照 GeneMapper ID-X 软件的格式要求,编写并导入 Panel 和 Bin 文件。 3. 大麻 STR 基因座复合扩增荧光检测体系的验证。根据 DNA 分析方法科 学工作组(Scientific Working Group on DNA Analysis Methods, SWGDAM)和国际法医遗传学会(International Society for Forensic Genetics, ISFG)的具体要求, 对构建的复合扩增荧光检测体系进行灵敏度、种属特异性、精确性、均衡性等法 医学评估,并对 126 份大麻样本进行群体遗传学参数调查。 【结果】1. 本研究从目前已开发的 33 个大麻 STR 基因座中筛选到 17 个符合法医学 应用的 STR 基因座,并选取 2 个大麻性别鉴定基因座,成功构建了包含 19 个基 因座的复合扩增荧光检测体系。 2. 本研究对构建的复合扩增荧光检测体系进行法医学验证,结果显示,当 模板 DNA 量低至 125 pg 时,仍然可以获得 19 个基因座上所有等位基因的完整 分型。种属特异性研究显示,非大麻源基因组 DNA 不会对大麻基因组 DNA 的 正确分型造成干扰。本研究构建的复合扩增体系中,每个等位基因都在其 Bin 范 围内,且所有基因座的平均 Stutter 比率均小于 15%。均衡性研究显示,该复合 扩增体系不同基因座杂合子平均均衡值在 0.8095(CS1)到 0.9516(4910)之间, 同一荧光标记基因座内平均均衡值在 0.6191(HEX)到 0.6809(TAMRA)之间, 不同荧光标记基因座间平均均衡值为 0.7072,该体系中 3 个均衡性参数的平均值 均满足推荐标准。 3. 本研究对构建的复合扩增荧光检测体系进行群体遗传学调查,结果显示, 17 个常染色体 STR 基因座在 126 份大麻样本中共观察到 181 个等位基因,其中 49 份毒品大麻均为雌性。17 个常染色体 STR 基因座在 126 份大麻样本中的杂合 度范围为 0.2381(1528)~ 0.7937(CS1),多态性信息含量范围为 0.2754(9269) ~ 0.9419(CS1),个体识别概率范围为 0.4624(9269)~ 0.9855(CS1),非父 排除概率范围为 0.0410(1528)~ 0.5873(CS1),累积个体识别概率大于 1-3.0×10-15, 累积非父排除概率大于 1-7.4×10-3。 【结论】本研究构建了包含 17 个常染色体 STR 基因座和 2 个性别鉴定基因座的大麻 复合扩增荧光检测体系,该体系具有灵敏度高、种属特异性好、准确性高等特点, 可用于大麻的种属鉴定、性别鉴定、个体识别及来源地推断等,同时,为司法鉴 定中大麻犯罪案件提供了新的技术手段。 |
其他摘要 | 【Objective】 1. To establish a fluorescent multiplex PCR system including 17 autosomal STR loci and 2 sex identification loci of Cannabis sativa. 2. To evaluate the effectiveness and forensic parameters of this system and validate that this system could meet the requirements for species identification, gender determination, individual identification and origin inference of Cannabis sativa. 【Methods】 1. Screening of Cannabis sativa STR loci. Developed Cannabis sativa STR loci were collected based on literature reports and GenBank database. Primer Premier 5.0 software and the Primer-BLAST primer design tool of the NCBI browser were used to design primers for these loci. Agarose gel electrophoresis was performed after the amplification of each single locus to verify the effect of primer design. 2. Establishment of a fluorescent multiplex PCR system of STR loci of Cannabis sativa. 17 autosomal STR loci and 2 sex identification loci were divided into four groups, and the 5'end of the forward primer of each locus was labeled with fluorescent dyes (FAM, HEX, TAMRA and ROX), respectively. By optimizing the final concentration of primers for each locus, as well as the multiplex amplification PCR reaction system and reaction conditions, a five-Dye fluorescence-labeled multiplex amplification detection system was constructed. According to the detection requirements of the 3500XL genetic analyzer, a 5-Dye spectrum correction file was established. According to the Sanger sequencing results of each locus, the alleles were nominated, and the Ladder were prepared. Finally, the Panel and Bin files were written and imported, according to the format requirements of the GeneMapper ID-X Software. 3. Validation of the fluorescent multiplex PCR system of STR loci of Cannabis sativa. According to the specific requirements of the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the International Society for Forensic Genetics (ISFG), forensic evaluation of the sensitivity, species specificity, accuracy and balance of the multiplex system were conducted, and the genetic parameters of 126 Cannabis sativa samples were calculated. 【Results】 1. In this study, 17 of the 33 previously used Cannabis sativa STR loci were selected for the requirements of forensic application. Together with 2 sex identification loci, a total of 19 loci were selected to successfully construct a multiplex amplification fluorescent detection system. 2. Forensic validation were performed on the constructed multiplex amplification fluorescent detection system. The results showed that complete profiles of all alleles can still be obtained even if the amount of template DNA was as low as 125 pg. Species-specific studies showed that non-Cannabis satiav-derived genomic DNA samples has little effect on the correct genotyping of Cannabis sativa genomic DNA. The multiplex amplification system constructed in this study could ensure that each allele was within its Bin range, and the average stutter ratio of each locus was less than 15%. The balance studies showed that the peak height ratios of heterozygous alleles per locus ranged from 0.8095 (CS1) to 0.9516 (4910), and the inter-loci balance with same fluorescent dyes ranged from 0.6191 (HEX) to 0.6809 (TAMRA), the inter-loci balance among different fluorescent labels was 0.7072, therefore, the average value of the three balance parameters of this system could meet the recommended standards. 3. In this study, the population investigation of Cannabis sativa was conducted using the constructed multiplex amplified fluorescent detection system, and the results showed that a total of 181 alleles were observed on the 17 autosomal STR loci from 126 Cannabis sativa samples. Among all of 126 samples, 49 marijuana samples were all females. The heterozygosity of the 17 autosomal STR loci in 126 Cannabis sativa samples ranged from 0.2381 (1528) to 0.7937 (CS1), the polymorphism information content (PIC) ranged from 0.2754 (9269) to 0.9419 (CS1), and the range of discrimination power was 0.4624 (9269) ~ 0.9855 (CS1), the range of excluding probalbility of paternity was 0.0410 (1528) ~ 0.5873 (CS1), the cumulative discrimination power was greater than 1-3.0×10-15, and the cumulative probability of exclusion was greater than 1-7.4×10-3. 【Conclusions】 This study constructed a multiplex amplification fluorescent detection system for the Cannabis sativa, containing 17 autosomal STR loci and 2 sex identification loci. This system has high sensitivity, good species specificity and high accuracy, which can be used for species identification, gender determination, individual identification and original inference of the Cannabis sativa, and providing a new technical method for Cannabis sativa criminal cases in for |
学号 | 181006038 |
发布年限 | 2021-06-23 |
毕业论文分类号 | 0R8009 |
原始专题 | 基础医学院 |
学位论文研究方向 | 法医物证学 |
参考文献 | 【Objective】 1. To establish a fluorescent multiplex PCR system including 17 autosomal STR loci and 2 sex identification loci of Cannabis sativa. 2. To evaluate the effectiveness and forensic parameters of this system and validate that this system could meet the requirements for species identification, gender determination, individual identification and origin inference of Cannabis sativa. 【Methods】 1. Screening of Cannabis sativa STR loci. Developed Cannabis sativa STR loci were collected based on literature reports and GenBank database. Primer Premier 5.0 software and the Primer-BLAST primer design tool of the NCBI browser were used to design primers for these loci. Agarose gel electrophoresis was performed after the amplification of each single locus to verify the effect of primer design. 2. Establishment of a fluorescent multiplex PCR system of STR loci of Cannabis sativa. 17 autosomal STR loci and 2 sex identification loci were divided into four groups, and the 5'end of the forward primer of each locus was labeled with fluorescent dyes (FAM, HEX, TAMRA and ROX), respectively. By optimizing the final concentration of primers for each locus, as well as the multiplex amplification PCR reaction system and reaction conditions, a five-Dye fluorescence-labeled multiplex amplification detection system was constructed. According to the detection requirements of the 3500XL genetic analyzer, a 5-Dye spectrum correction file was established. According to the Sanger sequencing results of each locus, the alleles were nominated, and the Ladder were prepared. Finally, the Panel and Bin files were written and imported, according to the format requirements of the GeneMapper ID-X Software. 3. Validation of the fluorescent multiplex PCR system of STR loci of Cannabis sativa. According to the specific requirements of the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the International Society for Forensic Genetics (ISFG), forensic evaluation of the sensitivity, species specificity, accuracy and balance of the multiplex system were conducted, and the genetic parameters of 126 Cannabis sativa samples were calculated. 【Results】 1. In this study, 17 of the 33 previously used Cannabis sativa STR loci were selected for the requirements of forensic application. Together with 2 sex identification loci, a total of 19 loci were selected to successfully construct a multiplex amplification fluorescent detection system. 2. Forensic validation were performed on the constructed multiplex amplification fluorescent detection system. The results showed that complete profiles of all alleles can still be obtained even if the amount of template DNA was as low as 125 pg. Species-specific studies showed that non-Cannabis satiav-derived genomic DNA samples has little effect on the correct genotyping of Cannabis sativa genomic DNA. The multiplex amplification system constructed in this study could ensure that each allele was within its Bin range, and the average stutter ratio of each locus was less than 15%. The balance studies showed that the peak height ratios of heterozygous alleles per locus ranged from 0.8095 (CS1) to 0.9516 (4910), and the inter-loci balance with same fluorescent dyes ranged from 0.6191 (HEX) to 0.6809 (TAMRA), the inter-loci balance among different fluorescent labels was 0.7072, therefore, the average value of the three balance parameters of this system could meet the recommended standards. 3. In this study, the population investigation of Cannabis sativa was conducted using the constructed multiplex amplified fluorescent detection system, and the results showed that a total of 181 alleles were observed on the 17 autosomal STR loci from 126 Cannabis sativa samples. Among all of 126 samples, 49 marijuana samples were all females. The heterozygosity of the 17 autosomal STR loci in 126 Cannabis sativa samples ranged from 0.2381 (1528) to 0.7937 (CS1), the polymorphism information content (PIC) ranged from 0.2754 (9269) to 0.9419 (CS1), and the range of discrimination power was 0.4624 (9269) ~ 0.9855 (CS1), the range of excluding probalbility of paternity was 0.0410 (1528) ~ 0.5873 (CS1), the cumulative discrimination power was greater than 1-3.0×10-15, and the cumulative probability of exclusion was greater than 1-7.4×10-3. 【Conclusions】 This study constructed a multiplex amplification fluorescent detection system for the Cannabis sativa, containing 17 autosomal STR loci and 2 sex identification loci. This system has high sensitivity, good species specificity and high accuracy, which can be used for species identification, gender determination, individual identification and original inference of the Cannabis sativa, and providing a new technical method for Cannabis sativa criminal cases in for |
全文文件名 | 181006038夏若成2021法医学.pdf |
文献类型 | 学位论文 |
条目标识符 | https://kms.wmu.edu.cn/handle/3ETUA0LF/178461 |
专题 | 温州医科大学 |
作者单位 | 溫州医科大学基础医学院 |
第一作者单位 | 温州医科大学 |
推荐引用方式 GB/T 7714 | 夏若成. 大麻STR基因座复合扩增体系的构建及法医学应用研究[D]. 温州医科大学,2021. |
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