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研究生:簡佳慶
研究生(外文):Chia-Ching Chien
論文名稱:建立基因晶片以檢測小鼠糞便中的傳染性病原
論文名稱(外文):Establishment of a gene chip for the detection of pathogens in mouse feces
指導教授:萬灼華陳慧文陳慧文引用關係
指導教授(外文):Cho-Hua WanHui-Wen Chen
口試委員:王明升闕玲玲王儷蒨
口試委員(外文):Ming-Hseng WangLing-Ling ChuehLih-Chiann Wang
口試日期:2014-06-06
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子暨比較病理生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:66
中文關鍵詞:基因晶片小鼠小病毒(MVM)小鼠濾過性小病毒(MPV)囓齒類螺旋桿菌小鼠諾羅病毒(MNV)多重引子聚合&;#37238;鏈鎖反應
外文關鍵詞:Gene chipMinute virus of miceMouse parvovirusrodent helicobactersMurine norovirusMultiplex PCR
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現今實驗小鼠族群中盛行率最高的傳染性疾病包含了小鼠諾羅病毒(murine norovirus, MNV)、濾過性小病毒(parvovirus)及螺旋桿菌(Helicobacter)等,且這些病原一旦汙染後通常不易將其由小鼠族群中清除。當實驗小鼠遭受感染後,其生理、免疫狀態可能受到改變、或導致研究數據受到影響,因此進行定期的健康監測是控制傳染疾病傳播的一項重要且必要的事情。許多近期的研究結果顯示,以傳統的衛兵鼠監測系統進行健康監測可能無法非常精確且有效地得知實驗小鼠的感染情形與實驗動物中心病原污染範圍的真正情況,其原因包含了衛兵鼠與病原的接觸時間不足、衛兵鼠對於某些傳染病原較不具感受性或因病原排出量較低而無法有效地感染衛兵鼠…等,因而有許多替代的檢測方式相繼被提出應用。為能夠直接應用於待檢測之目標小鼠的糞便檢體,並同時監測多種實驗小鼠傳染性病原,本研究選擇小鼠諾羅病毒(MNV)、小鼠小病毒(minute virus of mice, MVM)、小鼠濾過性小病毒(mouse parvovirus, MPV)、H. hepaticus, H. bilis以及H. typhlonius,嘗試建立一個多重引子聚合&;#37238;鏈鎖反應並配合專一性DNA探針的設計及基因晶片的原理,開發一個基因晶片診斷系統。此基因晶片診斷套組除了可精確地監測上述六種病原外,還能夠同時針對其他非MVM/非MPV的嚙齒類parvoviruses以及其他可能感染於實驗小鼠的helicobacters進行檢測。此基因晶片診斷系統為一高敏感性、高特異性之診斷套組,其針對所有目標病原之偵測極限皆能達到至少10 copies的高敏感性。此基因晶片診斷套組透過利用小鼠糞便檢體作為病原監測的目標,並且在不犧牲與不造成小鼠任何緊迫的人道前提下,能夠確實有效地應用於實驗小鼠的健康監測。

Murine norovirus, parvoviruses, and helicobacters are among the highly prevalent infectious pathogens in contemporary laboratory mice. Periodic health monitoring of laboratory animals is indispensable for disease control and prevention. Recent data suggests the sentinel health monitoring system may not efficiently and accurately demonstrate the infection status in laboratory animal colonies. In this study, a gene chip system was developed to simultaneously detect several infectious agents, including murine norovirus (MNV), minute virus of mice (MVM), mouse parvovirus (MPV), H. hepaticus, H. bilis, and H. typhlonius in mouse fecal samples. Besides these six specific pathogens, this assay could also monitor the existence of non-MVM/MPV rodent parvovirus and other helicobacters in feces and a mouse housekeeping gene. This gene chip assay is very sensitive and specific with detection limits of 10 copies for all target agents. The gene chip assay developed here could be a very useful tool to monitor infectious diseases of laboratory mice without sacrificing them or causing any stress/discomforts in animals.

目錄

口試委員審定書 i
誌謝 ii
摘要 iii
ABSTRACT iv
目錄 v
LIST OF TABLES vii
LIST OF FIGURES viii
第一章、序言 1
第一節、前言 1
第二節、實驗小鼠族群中高盛行率之傳染病原 1
第三節、小鼠濾過性小病毒(MVM, MPV) 3
第四節、螺旋桿菌(Helicobacter) 7
第五節、小鼠諾羅病毒(MNV) 10
第六節、實驗小鼠傳染病之診斷方式 13
第七節、研究動機與實驗設計 17
第二章、材料與方法 20
第一節、檢體來源 20
第二節、檢體之總核酸萃取 20
第三節、陽性控制組之建立 21
第四節、多重引子聚合&;#37238;鏈鎖反應之建立 22
第五節、基因晶片診斷套組之建立 25
第六節、基因晶片應用於小鼠糞便檢體中之病原檢測 29
第三章、結果 30
第一節、多重引子聚合&;#37238;鏈鎖反應之建立 30
第二節、基因晶片診斷套組之建立 32
第三節、基因晶片應用於小鼠糞便檢體中之病原檢測 34
第四章、討論 36
參考文獻 54

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