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研究生:陳俊憲
研究生(外文):Chun-Hsien Chen
論文名稱:常見麗蠅之快速鑑定及食屍性雙翅目研究
論文名稱(外文):The study of rapid identification of common blow fly species and necrophagous dipteran insect
指導教授:石正人石正人引用關係
指導教授(外文):Cheng-Jen Shih
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:61
中文關鍵詞:麗蠅快速鑑定食屍性雙翅目法醫昆蟲學
外文關鍵詞:blow flyrapid identificationnecrophagous dipteran insectforensic entomology
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麗蠅是台灣常見的食屍及食糞性昆蟲,廣泛分佈在全島各地,是法醫昆蟲學中最常被用來判斷死亡後至屍體被發現期間 (postmortem interval, PMI) 的昆蟲。然而,許多麗蠅的卵及幼蟲在外型十分相似,加上體型不大,單以外部形態鑑定較為費力。因此,如何在最短時間內,鑑定出屍體上麗蠅究竟屬於那個種類,成了法醫昆蟲學上相當重要的問題。本實驗以豬隻模擬人類屍體,採集豬屍體上常見之麗蠅,結果發現麗蠅中,以大頭金蠅 (Chrysomya megacephala)、紅顏裸金蠅 (C. rufifacies)、肥軀金蠅 (C. pinguis) 等族群最為龐大,為提供分子快速鑑定所需麗蠅之各生長期樣本,將豬屍體上所採得之雌蟲攜回實驗室,建立單雌培養之純系,再以第二代之雄性特徵分辨種類。以 DNA 作為分子標誌之研究中,嘗試以核糖體 DNA (rDNA)、粒線體 DNA (mtDNA) 及逢機增幅多態型 DNA (RAPD) 進行三種常見麗蠅之鑑別。在核糖體 DNA 研究上,增幅 rDNA 上的內轉錄區間 (internal transcribed spacers, ITS) 區域,以 ITSF01 及 ITS6 引子增幅 ITS1 片段,ITS3a 與 28S-2 引子增幅 ITS2;在 mtDNA 研究上,以引子 C1-J-1632 及 C1-N-2191 增幅mtDNA 上的細胞色素氧化次單位一 (cytochrome oxidase subunit I, COI) 部份區域。將增幅出之各 DNA 片段,進行限制片段長度多態型 (RFLP) 實驗,可清楚鑑別三種不同麗蠅。利用 genomic DNA,以 UBC (The University of British Columbia) 合成之 primer set 100/kit 進行 RAPD-PCR 實驗,結果共篩選出六組適用的 random primer,均可用以鑑別三種不同麗蠅。此外,觀察豬隻屍體的腐敗過程,發現主要可區分為新鮮期 (fresh stage)、腹部膨脹期 (bloated stage)、腐敗期 (decomposition stage)、殘骸期 (decay stage) 及乾屍期 (dry stage) 等五個階段,並比較各時期屍體溫度與環境溫度,發現屍溫通常較氣溫為高,其差異主要來自於麗蠅幼蟲的活動。量化各時期溫差之資料,配合已知麗蠅之生命表,可供未來法醫昆蟲學之應用。

Blow flies are the common insects around the island. It can provide an objective estimate for the postmortem interval (PMI). However, it is difficult to identify blow fly species, especially in immature stages (eggs, larvae and pupae), because their small size and morphological similarity. The identification of insect species occurs on carrion is therefore become a problem for forensic entomologists. In this study, we used pig as experimental material, which was killed and exposed on the field. Blow fly adults and larvae, which occurred on the surface of corpse were collected. Some of the larvae were reared in the laboratory with artificial diet to obtain pure colony. There were three commonly blow flies, Chrysomya megacephala, C. rufifacies and C. pinguis was found during the period of study. In DNA analysis, ribosomal DNA (rDNA), mitochondrial DNA (mtDNA) and random amplified polymorphic DNA (RAPD) were manipulated to distinguish blow fly species. The primer of ITSF01 and ITS6 were used to amplify rDNA ITS1 region, and primer of ITS3a, 28s-2 were used to amplify ITS2 region. C1-J-1632, C1-N-2191 were used to amplify mitochondrial DNA (partial gene of subunit I of cytochrome oxidase). All of the amplified products were digested with 9 restriction enzymes. The results of RFLP analysis revealed that ITS1 and partial COI region can be used to identify Chrysomya species. In the RAPD analysis, 100 random primers designed by UBC (The University of British Columbia) were tested. Only 6 primers can be used to distinguish three species of blow flies. In the field study of pig carrion decomposition process, there were five mainly stages were quantified: fresh stage, bloated stage, decomposition stage, decay stage and dry stage. We found that temperature in the carcass were higher than the air each stage of decomposition. It may be due to the heat produced by the maggots. The combination of these data with blow fly life table, could be applied to the forensic entomology in the estimation of PMI.

目錄 ----------------------------------------------------- i
中文摘要 ------------------------------------------------- iii
英文摘要 ------------------------------------------------- iv
壹、緒言 ------------------------------------------------- 1
貳、往昔研究 --------------------------------------------- 4
參、材料和方法 ------------------------------------------- 16
一、材料 ------------------------------------------------- 16
(一)、供試昆蟲 ------------------------------------------- 16
(二)、供試藥品 ------------------------------------------- 16
二、方法 ------------------------------------------------- 17
(一)、屍體上麗蠅之行為觀察 ------------------------------- 17
(二)、麗蠅 DNA 的萃取 ------------------------------------ 17
(三)、PCR 增幅目標 DNA ----------------------------------- 18
(四)、DNA 定序及序列分析 --------------------------------- 19
(五)、PCR-RFLP 分析 -------------------------------------- 19
(六)、RAPD-PCR 分析 -------------------------------------- 20
肆、結果與討論 ------------------------------------------- 21
一、食屍性雙翅目之研究 ----------------------------------- 21
(一)、豬屍體之腐敗過程 ----------------------------------- 21
(二)、屍體溫度與環境溫度之比較 --------------------------- 23
(二)、麗蠅之生態習性 ------------------------------------- 23
二、麗蠅之快速鑑定 --------------------------------------- 31
(一)、聚合連鎖反應-限制切割片段長度多態型分析 ------- 31
(二)、聚合連鎖反應-DNA 隨機增幅多態型分析 ------------- 43
伍、結論--------------------------------------------------- 46
陸、引用文獻 --------------------------------------------- 48
柒、附錄 ------------------------------------------------- 58
捌、誌謝 ------------------------------------------------- 61

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