【作者】 王国力；

【导师】 张伟； 孙启凡；

【作者基本信息】 山东大学 ， 生物工程（专业学位）， 2021， 硕士

【摘要】 分析鉴定案发现场遗留的生物检材可为案件侦查及现场重建提供重要线索。种属鉴定和组织属性鉴定是其中具有顺承关系的两个重要环节,均是法医物证鉴定需要解决的基本问题。由于现场检材的特殊性,许多传统鉴定方法被认为具有较大的局限性,这同时也强调了使用灵敏度高、特异性好、稳定性好的分子生物学鉴定方法的必要性。动物线粒体12S rRNA和16S rRNA基因因其多拷贝、种内遗传稳定性及种间差异性而被用于鉴定多种类型法医检材的种属来源;miRNA因其高稳定性和强大的组织特异性而被用于鉴定法医体液样本的组织属性来源。然而,目前报道的多种鉴定方法在实际应用中通常缺乏足够的方法普适性和结论一致性。为此,本研究分别从种属鉴定和体液鉴定两个层面进行新方法的探索,并为其可行性提供证据支持。在种属鉴定层面,本研究选取线粒体12S rRNA和16S rRNA基因,通过哺乳动物大类群中已有序列对比分析,确定序列高变区,在高变区两侧选取相对保守的区域分别设计出较短片段的通用引物。而后在部分高阶元代表性哺乳动物中进行PCR扩增和测序分析,通过RNA二级结构的预测和转换编码,将二级结构信息与核酸序列信息相结合识别物种。同时,基于二级结构的保守性,我们评估其在指导远缘物种序列同源比对方向的价值。结果表明:(1)特定短片段在研究对象内的PCR扩增成功率、测序成功率和物种识别率均为100%。(2)相较于常规比对方法,基于二级结构指导的序列比对可在一定程度上改善高分类阶元的序列比对结果和系统发育关系。(3)二级结构信息可增加用于系统发育分析的额外遗传信息,其中12S rRNA遗传信息总体增加23.37%,变异位点数增加20.69%,信息位点数增加20.86%;16S rRNA遗传信息总体增加17.95%,变异位点数增加约16.56%,信息位点数增加15.48%。(4)二级结构信息会提高部分支系的支持率,其中12S rRNA联合树中支持率升高的支系约占20%,支持率升高的幅度为2%-26%;16S rRNA联合树中支持率升高的支系约占13.64%,支持率升高的幅度为4%-10%。(5)基于二级结构信息的种属鉴定方法在人的种内鉴定上显示出一定的潜力。在体液鉴定层面,本研究通过使用近似绝对定量的标准曲线法,在包含五种体液的133份样本中建立了基于miR-451a和miR-21-5p拷贝数比值(451/21 Ratio)的月经血外周血检材鉴别方法,并对其进行了大量验证、评估及分析。结果表明:(1)在本研究构建的鉴别方法中,若0<451/21 Ratio<0.929,则体液样本类型为非血液;若0.929<451/21 Ratio<10.201,则体液样本类型为月经血;若451/21 Ratio>10.201,则体液样本类型为外周血。(2)62份月经血样本和24份外周血样本的外部验证准确率可达100%,且此鉴别方法不受样本的多种限制性因素(性别、年龄、月经血采集天数、暴露天数)制约。(3)此方法准确鉴别月经血与外周血的灵敏度可达0.2ng,其在不同实验平台及研究人员间血液来源鉴别的一致性可达100%,且此方法适用于月经血含量较高的混合血样鉴别。(4)月经血中451/21 Ratio的相对稳定具有一定的生物学意义,miR-451a和miR-21-5p可能共同靶向OSR1来作用于子宫内膜等生殖器官或组织,进而参与调节生殖系统的正常发育。本研究提出的种属鉴定和体液鉴定新方法兼具可行性和准确性,联合使用两种方法可以建立从科属种类到组织类型的综合鉴定体系,有望服务于法医实践。更多还原

【Abstract】 An accurate identification of biomaterials at crime scene can provide strong evidence for case investigation and crime reconstruction.Identification of biomaterials at the species level and the successive tissue level are the two important kinds of forensic identification.Due to the particularity of forensic biomaterials,traditional forensic identification always has limits,calling for the molecular biological methods with high sensitivity,specificity and stability.Mitochondrial 12S rRNA and 16S rRNA gene have been used for species identification in forensic materials because of their multiple copies,intraspecific genetic stability and interspecific differences.Meanwhile,miRNA has been used for tissue identification in body fluid samples by merits of their stability and specificity.However,these genetic markers and their applied methods are always lack of universality and consistency in practical application.In this study,we developed the novel methods for species identification and body fluid identification,and provided evidences to support their feasibility.In the part of species identification,the mitochondrial 12S rRNA and 16S rRNA genes were selected and studied in this study as follows.First,the most variable regions of the sequences were identified through the sequence comparation across a broad range of mammal groups.Then the relatively conservative regions which close to both sides of the high variation region were selected to design short universal primers.Second,PCR amplification and sequencing analyses were carried out in some representative mammals of higher taxonomic rank.The secondary structure information and nucleic acid sequence information were combined to resolve species after the prediction and conversion coding of RNA secondary structure.Meanwhile,based on the conservative feature of the secondary structure,we evaluated its value in guiding homology alignment of distant species.Species identification using 12S rRNA and 16S rRNA short regions showed that:(1)The success rate of PCR amplification,sequencing and species identification in this study were all 100%.(2)Compared with the conventional alignment methods,secondary-structure guiding alignment improved the sequence alignment and phylogenetic relationship in higher taxonomic rank to a certain extent.(3)Secondary-structure information increased the additional genetic information for phylogenetic analysis:the genetic information,variable characters and parsimony-informative characters of 12S rRNA increased by 23.37%,20.69%and 20.86%respectively,and 16S rRNA increased by 17.95%,16.56%and 15.48%respectively.(4)Secondary-structure information increased the support value of some clades.In the 12S rRNA sequence-structure combination tree,about 20%of the clades had increased support value,ranging from 2%to 26%;in the 16S rRNA sequence-structure combination tree,13.64%clades had increased support value,ranging from 4%to 10%.(5)The species identification strategy based on secondary-structure information showed a certain potential in the identification of the race of human species.In the part of body fluid identification,an identification strategy was established for distinguishing blood type between menstrual blood and peripheral blood based on the copy ratio of miR-451a and miR-21-5p(451/21 Ratio).This strategy was tested and validated by using the standard curve method of approximate absolute quantification in 133 samples that covered five types of body fluids.The results showed that:(1)If 451/21 Ratio was greater than 0 and less than 0.929,the sample should be non-blood materials;if 451/21 Ratio was greater than 0.929 and less than 10.201,the sample should be menstrual blood;if 451/21 Ratio was greater than 10.201,it should be peripheral blood.(2)The accuracy of external validations in 62 menstrual blood samples and 24 peripheral blood samples was 100%,and this strategy was not affected by gender,age,period of menstrual blood,and exposure days.(3)The minimum dose of menstrual and peripheral blood for this strategy was 0.2 ng,and the repeatability among different experimental platforms and researchers was 100%.Moreover,this strategy was also suitable for the identification of menstrual blood with small number of mixed blood samples.(4)The relative stability of the 451/21 Ratio in menstrual blood agree with gene functions,wherein miR-451a and miR-21-5p might bond the target OSR1 to act on the endometrium and other reproductive organs or tissues,and then participated in the regulation of the normal development of the reproductive system.The new strategies of species identification and body fluid identification developed in this study are feasible and accurate,and combination of the two strategies can establish a comprehensive identification system from family and genus to tissue type,which is promising in forensic practice.更多还原