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研究生:陳瑋芸
研究生(外文):Wei-Yun Chen
論文名稱:粒線體DNA細胞色素氧化酶次單元一(COI)之序列分析於大台北地區具法醫重要性麗蠅種類鑑定之應用
論文名稱(外文):Application of mitochondrial DNA Cytochrome c Oxidase subunit I (COI) sequence on identification of forensically important blow flie from Taipei area (Diptera: Calliphoridae)s
指導教授:蕭旭峰蕭旭峰引用關係洪挺軒
指導教授(外文):Shiuh-Feng ShiaoTing-Hsuan Hung
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:昆蟲學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:89
中文關鍵詞:麗蠅科分子鑑定粒線體DNA法醫昆蟲台北
外文關鍵詞:Calliphoridaemolecular identificationmitochondrial DNAforensic entomologyTaipei
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法醫昆蟲學 (forensic entomology) 是將昆蟲學知識應用作為於刑事案件調查中之一項證據,可提供調查人員判定死後間隔時間或命案之第一現場,由昆蟲所提供之科學證據可將死後間隔時間精確估算至幾小時。國內相關研究資料相當匱乏,本論文之研究目的即為建立台灣地區法醫昆蟲學之初步資料,期能提供後續實際應用及深入研究之參考。利用動物屍體之模擬實驗中麗蠅科昆蟲於屍體腐敗過程之早期即可發現,且其種類及數量均為屍體昆蟲相之冠,於大台北地區以自製陷阱進行具法醫重要性昆蟲之誘集,發現麗蠅科昆蟲為其中最優勢之一群,種類分別為大頭金蠅、紅顏金蠅、肥軀金蠅、銅綠蠅、紫綠蠅及瘦葉帶綠蠅等六種,同時將採集所得之幼蟲攜回實驗室內進行人工飼養並成功地建立包括大頭金蠅、肥軀金蠅、紅顏金蠅及紫綠蠅等四種之室內族群,此族群將可提供目前難以利用傳統形態分類法進行種類鑑定之麗蠅雌成蟲及幼期分析之用。取採集自大台北地區之六種麗蠅進行 DNA 分析,可發現不止雄成蟲,雌成蟲及幼期均可進行 DNA 分析,翅及蛹殼亦不例外;取飼養之大頭金蠅新鮮樣本雄成蟲以及各個時期之幼期,萃取基因組 DNA 以二倍連續稀釋後進行 PCR 反應所需最低 DNA 濃度之測定,可發現成蟲之頭部、胸部及腹部三處分節,幼期則以三齡幼蟲及蛹期 DNA 總量與可進行 PCR 反應之 DNA 稀釋倍數最大;取不同方法保存之麗蠅成蟲進行保存方法對 DNA 檢測影響之分析,由結果可發現以 75% 酒精保存與乾燥之樣本均可萃取相當量之基因組 DNA,而不同方法對 DNA 總量影響並無明顯差異,而保存時間越久,DNA 總量越低。取採集自大台北地區之六種麗蠅,及出雲山之海南綠蠅及南嶺綠蠅,萃取雄成蟲頭部之基因組 DNA,並以自行設計之引子進行粒線體 DNA COI 區域增幅並定序;以軟體分析序列並繪出系統樹,系統樹所得之群組關係與傳統形態分類方法所得之結果相符合;以序列分析結果設計之六種專一性引子,可成功地鑑別此八種麗蠅,由此簡單易辨之引子對將可提供法醫鑑識人員另一種快速且簡便之鑑定方法。
Forensic entomology is a discipline that apply entomological knowledge to assist criminal investigations, especially to estimate postmortem interval (PMI) or to locate the site where the death occurred. Although there were several forensic entomological research reports around the world, no related study has been done before in Taiwan. The aim of this study is to establish the first data of forensic entomology in Taiwan; and for further investigation and application, the forensically important insect fauna in Taipei area were surveyed by traps. The results showed that Calliphoridae is the major group. The dominant species are: Chrysomya megacephala, C. rufifacies, C. pinguis, Lucilia cuprina, L. porphyrina, and Hemipyrellia ligurriens. Four lab colonies of C. megacephala, C. rufifacies, C. pinguis, and L. porphyrina have successfully been established. PCR products of 1588 bp that amplified from different fragment (including head, thorax, abdomen, wings, and legs) of adults of C. megacephala, C. rufifacies, C. pinguis, L. cuprina, L. porphyrina and H. ligurriens, and different immature stages of C. megacephala, C. rufifacies, C. pinguis and L. porphyrina by using CCOI-F/CCOI-R primer pairs were yielded. For detecting the minimum template DNA concentration of the different adult body segments and different immature stages for PCR, 2-times sequential dilutions were performed. Results showed that the head, thorax, and abdomen in adults, 3rd instars and pupae have the highest dilution concentration. Analyzing the influence of different preserving methods to the genomic DNA contents by spectrophotometer showed no obvious difference between samples of different preserving methods; but in general, the longer the sample was preserved, the lesser the DNA can be extracted. The complete sequences of COI region of 12 blow fly populations of Taiwan and U.S.A. were aligned, and the phylogenetic analysis was performed by using PAUP*. Results showed that the phylogenetic relationships from molecular data highly agree with that from the morphological data. Six specific primer pairs were designed for differentiating eight blow fly species by PCR, and the results indicated the high resolution of using this primer key in practice. This result provided another quick and easy way for identifying blow fly species in forensic inspection from molecular level.
中文摘要
英文摘要
目錄
圖次
表次
附錄次
壹、前言
貳、往昔研究
一、法醫昆蟲學
二、麗蠅之分類地位、經濟重要性及於法醫昆蟲學之角色
三、分子分類
四、粒線體 DNA
五、分子標示於法醫昆蟲種類鑑定之應用
參、材料與方法
一、供試昆蟲採集
二、麗蠅之實驗室內族群建立及飼養
三、基因組 DNA 之萃取及PCR增幅反應
四、粒線體 DNA COI 片段之序列分析
(一) PCR 產物之純化
(二) 大腸桿菌之選殖
(三) 大腸桿菌質體 DNA 之萃取
(四) DNA 定序及序列分析
(五) 引子對之設計與 PCR 反應
肆、結果
一、大台北地區具法醫重要性之麗蠅採集
二、麗蠅之實驗室內族群建立及飼養
三、基因組 DNA 萃取及 PCR 增幅反應
四、麗蠅粒線體 DNA 細胞色素氧化酶次單元一 (Cytochrome c Oxidase subunit I, COI) 之序列分析
五、專一性引子對設計及分子檢索表建立
伍、討論
一、大台北地區具法醫重要性麗蠅採集及室內族群建立
二、分子鑑定法於具法醫重要性麗蠅種類鑑定之應用
三、具法醫重要性麗蠅 COI 區域序列分析、親緣關係建立與專一性引子設計
陸、參考文獻
柒、圖表
捌、誌謝
玖、附錄
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