线粒体COX1与12SrRNA及16SrRNA基因复合扩增种属鉴定研究

发布时间：2018-04-19 00:38

本文选题：种属鉴定 + 线粒体DNA　；参考：《山西医科大学》2013年硕士论文

【摘要】：目的：种属鉴定是法医物证检验的重要步骤,对现场提取的各种生物检材进行种属鉴定是进行其他鉴定的首要及关键环节。利用线粒体的特点,构建一种特异性好,灵敏度高,稳定性好,操作简便适用于法医学实践的扩增体系,以期解决当前法医种属鉴定所存在的问题及不足。方法：根据本课题的研究目的,探索复合扩增-琼脂糖凝胶电泳进行种属鉴定方法。参阅之前发表的种属鉴定方面的文章,确定筛选人类特异性基因和内参对照基因的原则,依照该原则并结合基因库中人及各种哺乳动物mtDNA序列,研究mt DNA的37个基因,并筛选出选出符合条件的编码基因,然后仔细比对,寻找异同,进而锁定目标基因序列；根据引物设计的原则,利用primer5及primer3(online)等多款引物设计软件,设计引物并优化；收集人和恒河猴,猪牛羊兔5种动物的肌肉或者血液样本,提取DNA,复合扩增线粒体12SrRNA,16SrRNA,COX1基因片段,摸索优化扩增条件,寻找特异性好、灵敏度高、结果稳定的扩增条件；扩增产物用琼脂糖凝胶电泳分离检测、254nm紫外灯下观察结果。结果：内参基因选择12SrRNA和16SrRNA,选择COX-1基因作为检测现代智人的特异性标志。设计四对引物12S1,12S2,16S和COX-1扩增相关区域。12S1和12S2引物扩增片段大小相同。电泳检测结果显示若16SrRNA、12SrRNA、 COX-1三个区域全部扩增出,显示三条带时,且大小分别为395bp、231bp、202bp判定样品来自人。对于非人类标本,包括猴在内的其它动物仅有16SrRNA、12SrRNA区的扩增条带。对于12SrRNA基因,我们在同一个扩增区域设计两对引物12S1和12S2,其中12S2扩增区,猪牛样品显示阳性,而12S1区域,除猪牛样品外,所有哺乳动物样品均可被扩增出。该扩增体系敏感度为2.5pg,特异性基因COX-1的灵敏度为10-8pg,因此适合对法医学上极微量检材的适用。结论：初步建立了可运用于法医学人类样品认定的复合扩增体系。该方法稳定性好、灵敏度高、特异性强、检测过程方便,适合法医学实践中的极微量检材的人类样品认定,为今后法医学种属鉴定提供了更为方便而实用的技术方法基础。
[Abstract]:Objective: species identification is an important step of forensic physical evidence examination, and the identification of species of various biological samples extracted in the field is the most important and key link of other identification.Based on the characteristics of mitochondria, an amplification system with good specificity, high sensitivity, good stability and easy operation was constructed to solve the problems and shortcomings in forensic identification.Methods: according to the purpose of this study, the method of multiplex amplification-agarose gel electrophoresis for species identification was explored.Referring to the previously published articles on species identification, the principles of screening human specific genes and internal reference control genes were determined. According to this principle, 37 genes of Mt DNA were studied in combination with the mtDNA sequences of human and various mammals in the gene bank.According to the principle of primer design, using several primer design software, such as primer5 and primer3online, the primers are designed and optimized.The muscle or blood samples of human, rhesus monkey, pig, sheep and rabbit were collected, and the DNA was extracted, the mitochondrial 12SrRNA-16SrRNA-COX1 gene fragment was amplified, and the optimized amplification conditions were explored to find the amplification conditions with good specificity, high sensitivity and stable results.The amplified products were separated by agarose gel electrophoresis and detected by UV lamp at 254 nm.Results: 12SrRNA and 16s rRNA were selected as the specific markers for the detection of modern Homo sapiens.Four pairs of primers, 12S1, 12S216s and COX-1, were designed to amplify the relevant region. 12S1 and 12S2 primers were the same size.The electrophoretic analysis showed that if the 16s rRNAs were 12s rRNAs, all the three regions of COX-1 were amplified, and the size of the three bands was 395bpn231bp20bp, respectively.For non-human specimens, there are only 16s rRNA 12s rRNA bands in other animals, including monkeys.For 12SrRNA gene, we designed two pairs of primers 12S1 and 12S2 in the same amplification region, in which 12S2 amplification region, porcine and bovine samples showed positive, and 12S1 region, all mammalian samples except porcine cattle samples could be amplified.The sensitivity of the amplification system was 2.5 PG and the sensitivity of specific gene COX-1 was 10-8 PG.Conclusion: a multiplex amplification system which can be applied to the identification of forensic human samples was established.This method has the advantages of good stability, high sensitivity, high specificity and convenient detection process. It is suitable for the identification of human samples in forensic practice and provides a more convenient and practical technical basis for the identification of forensic medicine species in the future.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：D919.4

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