本文選題：法醫(yī)昆蟲學(xué) + 線粒體DNA��； 參考：《中南大學(xué)》2011年碩士論文

【摘要】：背景： 法醫(yī)昆蟲學(xué)(Forensic entomology)是一門應(yīng)用昆蟲學(xué)知識(shí)解決法律上問題尤其是刑事案件偵破的新興學(xué)科,主要用于腐敗尸體的死后間隔時(shí)間(Postmortem Interval, PMI)的推斷。主要的嗜尸性昆蟲包括雙翅目的蠅類和鞘翅目的甲蟲等。其中,雙翅目中的嗜尸性蠅類是研究和實(shí)際應(yīng)用最多的類型,它們以腐敗尸體為食繁殖生長(zhǎng)的特殊生活習(xí)性常常為兇殺案或無名尸案?jìng)善铺峁┯欣�索。 機(jī)體死后不同階段,到達(dá)尸體上的嗜尸性蠅類種類不同,呈現(xiàn)出較強(qiáng)的演替現(xiàn)象。嗜尸性麗蠅(blow fly)是最早在尸體上出現(xiàn)的蠅類,其中麗蠅對(duì)尸體敏感性最強(qiáng),往往最先到達(dá)尸體,并且在死亡后幾小時(shí)之內(nèi)產(chǎn)卵于尸體上,所以,測(cè)定尸體上嗜尸性蠅類的種類及其發(fā)育齡期往往是估計(jì)死亡后時(shí)間(PMI)的關(guān)鍵昆蟲資料。 早中期尸體上的昆蟲的大部分處于非成熟階段,昆蟲的卵與早期幼蟲在形態(tài)上非常相似,因此使用傳統(tǒng)形態(tài)學(xué)方法鑒別比較困難。為了解決這個(gè)問題,本研究使用了近些年來發(fā)展迅速的分子生物學(xué)技術(shù),運(yùn)用自行設(shè)計(jì)的16SrDNA引物從基因水平探索昆蟲種類鑒別提供基礎(chǔ)資料。 目的： 本研究根據(jù)文獻(xiàn)報(bào)道資料,自行設(shè)計(jì)可以獲取短片段16SRibosomal DNA (16S rDNA)序列的引物,利用短片段16SrDNA區(qū)分常見嗜尸性麗蠅,尋找常見嗜尸性麗蠅的分子標(biāo)記,為利用嗜尸性麗蠅推斷死亡時(shí)間提供技術(shù)支持。 方法： 隨機(jī)采集我國(guó)多個(gè)地區(qū)室外多點(diǎn)放置動(dòng)物尸體上的蠅類樣本。采用改進(jìn)的小型昆蟲DNA勻漿方法提取上述蠅類mtDNA,蛋白核酸測(cè)定儀檢測(cè)DNA純度及濃度,Eppendorf5331梯度PCR儀進(jìn)行擴(kuò)增；7%聚丙烯酰胺非變性凝膠連續(xù)緩沖體系垂直電泳和0.8%瓊脂糖凝膠電泳檢測(cè)線粒體DNA及PCR產(chǎn)物,PCR膠回收試劑盒純化及PCR產(chǎn)物序列測(cè)定；MEGA4軟件包進(jìn)行序列分析和構(gòu)建系統(tǒng)發(fā)育樹。 結(jié)果： 采用對(duì)線粒體16SrDNA基因進(jìn)行PCR擴(kuò)增和DNA測(cè)序的方法,有效地檢測(cè)出嗜尸性麗蠅樣本的基因序列。通過分子進(jìn)化關(guān)系分析,得到各樣本的親緣關(guān)系圖,從而有效的鑒定出4屬(裸金蠅屬、金蠅屬、綠蠅屬、麗蠅屬),10種蠅類(絲光綠蠅、銅綠蠅、叉葉綠蠅、亮綠蠅、紫綠蠅、南嶺綠蠅、大頭金蠅、緋顏裸金蠅、紅頭麗蠅、反吐麗蠅)的種屬關(guān)系。 結(jié)論： 1. mtDNA上16SrDNA中289bp基因序列分析是鑒定麗蠅科種類一個(gè)有效工具。該檢測(cè)方法快速、簡(jiǎn)便和精確,可以作為法醫(yī)學(xué)鑒別嗜尸性蠅類種類提供新的方法,從而為死亡時(shí)間推斷提供依據(jù)。 2.短片段16SrDNA序列存在著一定的局限性,緋顏裸金蠅在UPGMA系統(tǒng)發(fā)育樹中單獨(dú)成一簇,與同為金蠅屬的大頭金蠅分開,因此使用分子標(biāo)記技術(shù)鑒別蠅的種類只是對(duì)傳統(tǒng)形態(tài)學(xué)方法的一個(gè)補(bǔ)充。 3.同一種蠅類在不同地區(qū)的差異性不大,需要加大同一種類在不同地區(qū)的樣本量進(jìn)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 .

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：D919.1

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本文編號(hào)：1751139