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研究生:王克偉
研究生(外文):Ko-Wei Wang
論文名稱:兩種小鼠濾過性小病毒多重引子聚合酶鏈鎖反應之建立與台灣實驗小鼠感染之調查
論文名稱(外文):Development of the Multiplex PCR Assays for Mouse Parvovirus (MPV) and Minute Virus of Mice (MVM), and the Surveillance of MPV and MVM Infection in Laboratory Mice in Taiwan
指導教授:萬灼華
指導教授(外文):Cho-Hua Wan
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
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:2010
畢業學年度:98
語文別:中文
論文頁數:75
中文關鍵詞:小鼠濾過性小病毒 (MPV)小鼠小病毒 (MVM)小鼠的濾過性小病毒多重引子聚合酶鏈鎖反應台灣小鼠的濾過性小病毒感染率
外文關鍵詞:mouse parvovirusminute virus of miceMPV/MVM multiplex PCRMPV prevalenceMVM prevalence
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Minute virus of mice (MVM) 與mouse parvovirus (MPV) 兩種小鼠的濾過性小病毒 (mouse parvoviruses) 為實驗小鼠中常見的病原。此病毒常經由污染的試劑與細胞培養液在實驗操作下或經帶原小鼠傳染給實驗動物中心的實驗小鼠。多項研究資料指出小鼠的濾過性小病毒會影響in vitro與 in vivo的免疫、腫瘤以及移植等相關的研究結果。為同時偵測此兩種病毒並顯示檢體樣本DNA品質,本研究針對MPV與MVM基因序列與小鼠的管家基因 (housekeeping gene),建立了一個多重引子聚合酶鏈鎖反應試劑 (MPV/MVM/Actin Multiplex PCR Assay)。利用此試劑可特異性的增幅出MPV與MVM病毒基因,但不會複製其他的rodent parvoviruses的基因;針對敏感度測試結果顯示本試劑可同時偵測到50個MVM與MPV病毒,此結果顯示本診斷試劑具有高特異性與高敏感度。在模擬MPV與MVM不等量共同感染,即使兩種病毒量相差高達200倍的情況下,本試劑仍維持至少可偵測到50個病毒的敏感度,顯示本試劑偵測效果穩定。利用本試劑於台灣實驗小鼠的濾過性小病毒感染調查中,分別由7個單位蒐集到14種品系,共174隻實驗小鼠,其中MPV的陽性率高達11.5%,但未偵測到MVM的感染。此結果顯示,MPV在台灣實驗小鼠的感染率高,為了維持動物實驗數據的正確與品質,建議應將此病毒列入主要的疾病監控項目。此MPV/MVM/Actin Multiplex PCR Assay可同時且有效的偵測到MPV與MVM兩種病毒,亦可應用於監測其他生物製劑的污染情形,未來可成為實驗動物疾病管理上的一個利器。

Mouse parvoviruses are among the highly prevalent infectious pathogens in contemporary mouse colonies. There are two serotypes, minute virus of mice (MVM) and mouse parvovirus (MPV). Mouse parvoviruses have a predilection for mitotically active cells and can interfere with immunology, transplantation, and oncology research through virus-infected rodents, contaminated cells and biological materials. In order to detect these two viruses simultaneously, a multiplex PCR assay that amplifies the VP gene of MPV and MVM, and a mouse housekeeping gene has been developed in this study. The MPV/MVM/Actin Multiplex PCR assay specifically detects MPV and MVM, but doesn’t amplify KRV, RMV-1, RPV-1c, and H-1. The multiplex PCR assay could simultaneously detect both MVM and MPV in as low as 50 copies in the condition of equal-amount dual infection. The sensitivity of this multiplex PCR assay remained to detect at least 50 copies of MPV when the copies of MVM were 200 times higher than the MPV copy number, and vice versa. The prevalence surveillance result revealed that 3 out of 7 laboraotry mouse colonies were contaminated with MPV with the prevalence of 11.5%; however, none of the tested mouse colony was contaminated with MVM. The MPV/MVM/Actin Multiplex PCR assay developed in this study can provide a useful tool in the mouse health monitoring in the future.

目錄
致謝 I
中文摘要 III
Abstract IV
目錄 V
Table VII
Figure VIII
第一章、序論 1
第一節、前言 1
第二節、小鼠的濾過性小病毒 (Mouse parvoviruses) 簡介 2
第三節、小鼠的濾過性小病毒 (Mouse parvoviruses) 感染 5
第四節、小鼠的濾過性小病毒 (Mouse parvoviruses) 對研究的影響 8
第五節、流行病學 11
第六節、診斷方式 12
第二章、研究動機與實驗設計 17
第三章、材料與方法 18
第一節、實驗設計與流程 18
第二節、實驗檢體與細胞來源 18
第三節、聚合酶鏈鎖反應陽性控制組之建立 21
第四節、MPV與MVM聚合酶鏈鎖反應試劑開發 25
第五節、台灣實驗小鼠與細胞培養液的濾過性小病毒感染調查 31
第四章、結果 33
第一節、實驗檢體與細胞培養液收集 33
第二節、MPV與MVM聚合酶鏈鎖反應試劑之開發 33
第三節、台灣實驗小鼠與細胞培養液的濾過性小病毒感染調查 37

第五章、討論 39
第一節、MPV/MVM聚合酶鏈鎖反應試劑之開發 40
第二節、台灣實驗小鼠MPV與MVM病毒之監測 43
第三節、細胞培養液中MPV與MVM病毒之監測 45
第四節、疾病控制策略 46
第五節、結論 47
第六節、未來工作與展望 47
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