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研究生:陳軒振
研究生(外文):Hsuan-Chen Chen
論文名稱:即時反轉錄恆溫環形核酸擴增法應用於精液鑑定之研究
論文名稱(外文):The study of real-time reverse transcription loop-mediated isothermal amplification for semen identification
指導教授:謝幸媚謝幸媚引用關係
指導教授(外文):Hsing-Mei Hsieh
學位類別:碩士
校院名稱:中央警察大學
系所名稱:鑑識科學研究所
學門:軍警國防安全學門
學類:警政學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:98
中文關鍵詞:體液鑑定反轉錄恆溫環形核酸擴增法KLK3基因RGS22基因SPATA32基因限制酶切割多重引子恆溫環形核酸擴增法
外文關鍵詞:Body fluid identificationRT-LAMPKLK3RGS22SPATA32restriction enzyme digestionmultiplex-LAMP
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自2000年恆溫環形核酸擴增法( LAMP )問世後,許多領域均善用其可在恆溫環境中短時間大量擴增核酸之特點,發展一連串簡易快速具高靈敏度之分析系統,惟其仍未在鑑識領域有所應用。而體液鑑定之重要性在刑案偵查中逐受重視,因此本研究欲結合兩者,發展以反轉錄恆溫環形核酸擴增法( RT-LAMP )進行體液鑑定之系統,且以性侵害案件中常見之精液檢體為標的進行測試。經由文獻探討及NCBI資料庫所記載之精液表現特異性基因中,共篩選出五個基因位,在經個體重複性及穩定度試驗後發現,KLK3、RGS22及SPATA32三個基因在精液中具穩定表現之結果。惟特異性部分,除了精液之外,RGS22及SPATA32在部分個體之周邊血及陰道分泌物中亦會有表現,KLK3則具有精液特異性。為偵測及確認LAMP產物,本研究利用即時濁度儀、洋菜膠電泳及限制酶切割作用等技術進行分析,並比較不同技術偵測LAMP產物之結果。結果中顯示,部分個體之尿液在電泳圖中呈現與精液產物不同之連續亮帶分布,經限制酶作用後,可確認其為非特異性產物。本研究中為同時分析18S rRNA (內控制組)與具精液表現特異性之KLK3兩個基因位之表現情形,以節省時間與檢體所需量,遂發展multiplex RT-LAMP技術,並利用不同螢光之呈現來研判檢體是否為精液。此外,本研究中亦嘗試發展在未萃取RNA狀態下,直接以體液經簡易前處理方式後即進行RT-LAMP反應,以提升檢體鑑定之時效性。經稀釋之精液,在添加DTT及加熱100°C反應十分鐘之條件中,均能成功擴增出上述三個基因之標的片段。綜合上述,目前所篩選之基因中僅KLK3具有特異性及良好穩定性之表現。在與18S rRNA組合之多重引子系統中亦能針對精液得到明確之螢光結果。本研究所發展之系統,若能針對其特異性進行改善,將可提升其於刑事鑑識上應用之價值。
Loop-mediated Isothermal Amplification, LAMP, has been developed since 2000. It can amplify nucleic acids in short time with isothermal condition, therefore, a series of analytical systems with high sensitivity are developed in many fields. However, it has not been used in forensic science. Due to the body fluid identification has become more valuable in forensic science, in this study, we focus on the combination of these two topics and development of a system by using the reverse transcription loop-mediated isothermal amplification (RT-LAMP) to identify semen, the most important body fluid in sexual assault cases. According to the references and NCBI database, five gene loci were selected to test by using the system. Stable expressions in semen samples were observed for three genes of them. The results also showed that the KLK3 was with semen specificity, and the RGS22 and SPATA32 with low semen specificity because some samples from blood and vaginal fluids also showed the KLK3 expression. In this study, different methods including the real-time turbidimeter test and gel electrophoresis were used to detect the LAMP products; and the restriction enzyme digestion was further used to confirm them. By using this method, LAMP products from some urine samples were confirmed to be non-specific. The multiplex-LAMP system for combination of 18S rRNA and KLK3 were developed and the products were detected by using different colors of fluorescence. The multiplex system can reduce labor and DNA template required. For direct LAMP without RNA extraction, the dilution semen sample with DTT was heated at 100°C for 10 mins and then the RT-LAMP was performed. The results showed that all the tested genes can be amplified successfully. In conclusion, improvement of the specificity of our system developed in this study will enhance its values on forensic applications.
謝誌 Ⅰ
中文摘要 Ⅱ
Abstract Ⅳ
目錄 Ⅵ
表目錄 Ⅸ
圖目錄 Ⅹ
第一章 前言 1
第二章 文獻探討 4
第一節 精液鑑定方法 4
第二節 RNA鑑定於目前刑事鑑識相關之研究 5
第三節 針對精液具特異性之基因介紹 6
一、SEMG1 (Semenogeline 1)基因 6
二、KLK3 (Kallikrein related peptidase 3)基因 7
三、PRM1 (Protamine 1)基因 8
四、RGS22基因 9
五、SPATA32基因 9
第四節 恆溫環形核酸擴增法簡介 10
一、恆溫環形核酸擴增法原理 10
二、偵測方法 13
三、相關應用研究 18
第五節 多重引子恆溫環形核酸擴增法之發展與應用 19
第三章 實驗材料與方法 22
第一節 研究流程 22
第二節 檢體來源 24
第三節 實驗方法 25
一、RNA萃取 25
二、LAMP引子設計 26
三、RT-LAMP反應 27
四、RT-LAMP產物偵測與確認 27
五、Multiplex LAMP 29
六、Direct LAMP 31
第四章 結果與討論 32
第一節 LAMP引子測試結果 32
第二節 精液標記之特異性測試 37
一、KLK3 38
二、RGS22 47
三、SPATA32 59
第三節 Multiplex LAMP測試結果 69
第四節 Direct LAMP測試結果 72
第五章 結論 74
第六章 未來展望 79
第七章 參考文獻 81
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