16SrDNA对中国常见嗜尸性丽蝇的种类鉴定及法医学意义

发布时间：2018-04-14 21:46

本文选题：法医昆虫学 + 线粒体DNA　；参考：《中南大学》2011年硕士论文

【摘要】：背景：法医昆虫学(Forensic entomology)是一门应用昆虫学知识解决法律上问题尤其是刑事案件侦破的新兴学科,主要用于腐败尸体的死后间隔时间(Postmortem Interval, PMI)的推断。主要的嗜尸性昆虫包括双翅目的蝇类和鞘翅目的甲虫等。其中,双翅目中的嗜尸性蝇类是研究和实际应用最多的类型,它们以腐败尸体为食繁殖生长的特殊生活习性常常为凶杀案或无名尸案侦破提供有利线索。机体死后不同阶段,到达尸体上的嗜尸性蝇类种类不同,呈现出较强的演替现象。嗜尸性丽蝇(blow fly)是最早在尸体上出现的蝇类,其中丽蝇对尸体敏感性最强,往往最先到达尸体,并且在死亡后几小时之内产卵于尸体上,所以,测定尸体上嗜尸性蝇类的种类及其发育龄期往往是估计死亡后时间(PMI)的关键昆虫资料。早中期尸体上的昆虫的大部分处于非成熟阶段,昆虫的卵与早期幼虫在形态上非常相似,因此使用传统形态学方法鉴别比较困难。为了解决这个问题,本研究使用了近些年来发展迅速的分子生物学技术,运用自行设计的16SrDNA引物从基因水平探索昆虫种类鉴别提供基础资料。目的：本研究根据文献报道资料,自行设计可以获取短片段16SRibosomal DNA (16S rDNA)序列的引物,利用短片段16SrDNA区分常见嗜尸性丽蝇,寻找常见嗜尸性丽蝇的分子标记,为利用嗜尸性丽蝇推断死亡时间提供技术支持。方法：随机采集我国多个地区室外多点放置动物尸体上的蝇类样本。采用改进的小型昆虫DNA匀浆方法提取上述蝇类mtDNA,蛋白核酸测定仪检测DNA纯度及浓度,Eppendorf5331梯度PCR仪进行扩增；7%聚丙烯酰胺非变性凝胶连续缓冲体系垂直电泳和0.8%琼脂糖凝胶电泳检测线粒体DNA及PCR产物,PCR胶回收试剂盒纯化及PCR产物序列测定；MEGA4软件包进行序列分析和构建系统发育树。结果：采用对线粒体16SrDNA基因进行PCR扩增和DNA测序的方法,有效地检测出嗜尸性丽蝇样本的基因序列。通过分子进化关系分析,得到各样本的亲缘关系图,从而有效的鉴定出4属(裸金蝇属、金蝇属、绿蝇属、丽蝇属),10种蝇类(丝光绿蝇、铜绿蝇、叉叶绿蝇、亮绿蝇、紫绿蝇、南岭绿蝇、大头金蝇、绯颜裸金蝇、红头丽蝇、反吐丽蝇)的种属关系。结论： 1. mtDNA上16SrDNA中289bp基因序列分析是鉴定丽蝇科种类一个有效工具。该检测方法快速、简便和精确,可以作为法医学鉴别嗜尸性蝇类种类提供新的方法,从而为死亡时间推断提供依据。 2.短片段16SrDNA序列存在着一定的局限性,绯颜裸金蝇在UPGMA系统发育树中单独成一簇,与同为金蝇属的大头金蝇分开,因此使用分子标记技术鉴别蝇的种类只是对传统形态学方法的一个补充。 3.同一种蝇类在不同地区的差异性不大,需要加大同一种类在不同地区的样本量进一步研究。
[Abstract]:Background :

It is a newly developed subject that uses the knowledge of Kunming to solve the legal problems , especially the detection of criminal cases , which is mainly used to deduce the dead time interval ( PMI ) of the dead bodies .

In different stages of body death , the species of corpse flies on the body are different and show a strong succession . The flies are the earliest flies that appear on the body , and the flies have the strongest sensitivity to the body , often first arrive at the corpse , and lay eggs on the body within a few hours after death . Therefore , it is often the key insect data of estimating the post - death time ( PMI ) .

In order to solve the problem , the present study has used the molecular biology techniques developed rapidly in recent years , and uses 16SrDNA primers designed by ourselves to explore the identification of insect species from the gene level .

Purpose :

According to the data reported in the literature , a primer of 16SRibosomal DNA ( 16S rDNA ) sequence can be obtained by self - design . Using short - segment 16SrDNA to distinguish the common female flies , the molecular markers of common necrophagous flies were found .

Method :

An improved small insect DNA homogenization method was used to extract the mtDNA of the flies . The purity and concentration of DNA were determined by using an Eppendorf5331 gradient PCR instrument .
7 % polyacrylamide non - denaturing gel continuous buffer system vertical electrophoresis and 0.8 % agarose gel electrophoresis to detect mitochondrial DNA and PCR products , PCR glue recovery kit purification and PCR product sequence determination ;
MEGA4 software package carries out sequence analysis and builds phylogenetic trees .

Results :

Using PCR and DNA sequencing of mitochondrial 16SrDNA , the genetic sequences of the samples were detected by PCR and DNA sequencing . The phylogenetic relationships of each sample were obtained .

Conclusion :

1 . The analysis of the 289bp gene in 16SrDNA from mtDNA is an effective tool for identifying the species of Liriomyza . The detection method is rapid , simple and accurate , and can provide a new method for the identification of the species of flies in forensic medicine , so as to provide the basis for the estimation of death time .

2 . There are some limitations in the sequence of 16SrDNA in the short segment , and in the development tree of UPGMA , there is a single cluster in the development tree of UPGMA , and the species of flies are only complementary to the traditional morphological method using molecular marker techniques .

3 . The diversity of the same species of flies in different regions is not large , and the sample size of the same species in different regions needs to be increased further .

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：D919.1

【参考文献】