劉振軒臺灣大學:獸醫學研究所蔡承龍Tsai, Cheng-LongCheng-LongTsai2010-05-042018-07-092010-05-042018-07-092009U0001-3007200916422000http://ntur.lib.ntu.edu.tw//handle/246246/178960Malignant catarrhal fever (MCF) is a fatal viral disease of ruminants. There are four viruses to cause this disease. Ovine herpesvirus 2 is one of them to cause the worst danger. Because of the disease involved subclinical carriers with virus shedding and dead-end hosts, it is hard to eradicate via inspection and quarantine. Owing to false positive and false negative, CI-ELISA is usually to screen the herd for understanding the MCF infection, not for diagnosis. The nested PCR is the most common assay used for diagnosing MCF. It is highly sensitive and specific, but time consuming. A novel DNA amplification method, loop-mediated isothermal amplification (LAMP), can amplify DNA with high specificity and rapidity under isothermal conditions and can be employed in the field test. The aim of this study is to develop the LAMP method for diagnosing MCF and compare the results among CI-ELISA, LAMP and nested PCR. A total of 107 samples of archived paraffin samples and peripheral blood lymphocytes from MCF positive and unknown herds were obtained for this study. The result of the CI-ELISA, nested PCR and LAMP assays show positive rate of 67.5％, 37.5％ and 45％, respectively, in MCF-positive herds. The results were all negative by the established assay in nondetected herds. In conclusion, the LAMP method is about six times faster than the nested PCR in the diagnosis of MCF. It takes approximately one hour to obtain the results and has similar sensitivity with nested PCR. This technique provides highly potential for rapid and specific diagnosis of MCF.目次謝 i要 iibstract iii次 v次 viii次 ix一章 實驗動機與目的 1二章 文獻探討 3一節 惡性卡他熱 （Malignant catarrhal fever, MCF) 3. 1. 1 MCF 之歷史背景、宿主與流行病學 3. 1. 2 MCF 的臨床表徵 5. 1. 3 MCF 之病毒特性 6. 1. 4 MCF 的研究現況 9. 1. 5 MCF 的診斷方法 12. 1. 6 MCF 在台灣之感染現況 13二節 競爭抑制型酵素連結免疫吸附法（Competitive inhibition enzyme-linked immunosorbent assay, CI-ELISA）與巢式聚合酶連鎖反應（nested polymerase chain reaction, nested PCR） 14三節 迴路媒介恆溫增幅法 （Loop-mediated isothermal amplification assay, LAMP） 16. 3. 1 LAMP 技術之發展與研究現況 16. 3. 2 LAMP 技術之增幅原理 17. 3. 3 LAMP 技術之產物檢測 19. 3. 4 LAMP 技術之應用 20三章 材料與方法 22一節 實驗設計與流程 22二節 檢體來源 23三節 競爭抑制型酵素結合免疫吸附法 （competitive inhibition enzyme-linked immunosobent assay, CI-ELISA）檢測 23四節 巢式聚合酶連鎖反應（nested PCR）檢測 25. 4. 1 檢體收集 25. 4. 2 蠟塊組織中病毒核酸之前處理與抽取 25. 4. 3 周邊血液淋巴球中病毒核酸之前處理與抽取 26. 4. 4 巢氏聚合酶連鎖反應（nested polymerase chain reaction, nested PCR） 27五節 LAMP 技術之建立 30. 5. 1 LAMP 引子對之設計 30. 5. 2 LAMP 反應條件之建立 31. 5. 3 LAMP 與 nested PCR 反應之比較 33. 5. 4 LAMP 應用於臨床檢體 33四章 結果 34一節 CI-ELISA 之檢測結果 34. 1. 1 CI-ELISA 之％ inhibition值建立 34. 1. 2 臨床檢體之 CI-ELISA 檢驗結果 34二節 分子生物學檢測 35. 2. 1 以 nested PCR 檢測之結果 35. 2. 2 Nested PCR 產物定序 35三節 LAMP 技術之建立 35. 3. 1 LAMP 技術引子對之選擇 35. 3. 2 LAMP 技術之反應條件最佳化 36. 3. 3 LAMP 與 nested PCR 反應之比較結果 37. 3. 4 臨床檢體檢驗結果之分析 37五章 討論 39一節 CI-ELISA 檢驗結果之探討 39二節 Nested PCR 檢驗結果之探討 39. 2. 1 Nested PCR 之建立 39. 2. 2 Nested PCR 之分析 41三節 LAMP 法之建立 41. 3. 1 設計 LAMP 引子對 41. 3. 2 建立 LAMP 法最適當之反應條件 42. 3. 3 觀察方法 43. 3. 4 比較 nested PCR 與 LAMP 之敏感度 44. 3. 5 分析比較 CI-ELISA、nested PCR 與 LAMP 之檢驗結果 44. 3. 6 LAMP法的應用與限制 47四節 結論 48考文獻 49application/pdf2768158 bytesapplication/pdfen-US惡性卡他熱綿羊第二型疱疹病毒競爭抑制型酵素結合免疫吸附法巢式聚合酶連鎖反應迴路媒介恆溫增幅法Malignant catarrhal fever (MCF)Ovine herpesvirus 2 (OvHV-2)Competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA)Nested polymerase chain reaction (nested PCR)Lood-mediated isothermal amplification (LAMP)thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178960/1/ntu-98-R96629015-1.pdf