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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46857完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 龐飛 | |
| dc.contributor.author | Chun-Ni Hsiao | en |
| dc.contributor.author | 蕭君倪 | zh_TW |
| dc.date.accessioned | 2021-06-15T05:42:18Z | - |
| dc.date.available | 2011-08-21 | |
| dc.date.copyright | 2010-08-21 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-20 | |
| dc.identifier.citation | Albina, E., 1997, Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview. Vet Microbiol55, 309-316.
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Vet Microbiol126, 1-10. 杜啟華, 賴秀穗, 2006, Investigation of the Causes of Post-weaning Multisystemic Wasting Syndrome and Phylogenetic Analysis of Porcine Circovirus Type II isolates. 邱明堂, 蘇保誠, 莊孟修, 林昭男, 2004, 台灣地區罹病豬隻中第二型豬環狀病毒感染狀況之調查. 台灣獸醫誌30, 163-168. 張志堅, 潘居祥, 李淑慧, 黃天祥, 王群, 鍾明華, 2007, 臺灣養豬場豬隻呼吸道疾病常見病原之分析. 家畜衛試所研報42, 71-78. 黃天祥, 羣, 王., 鄧明中, 張志堅, 李淑慧, 潘居祥, 李敏旭, 鍾明華, 2009, 2008年送檢豬隻病例病毒檢出率及疫情分析. 家畜衛試所研報44, 35-44. 楊程堯, 張聰洲, 林昭男, 蔡敬屏, 邱明堂, 2007, 台灣地區豬呼吸道疾病綜合症相關病原之分析. 臺灣獸醫誌33, 40-46. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46857 | - |
| dc.description.abstract | Porcine circovirus (PCV2)、porcine reproductive and respiratory syndrome virus (PRRSV)、swine influenza virus (SIV)、Torque teno virus (TTV)、porcine parvovirus (PPV) 以及classical swine fever virus (CSFV)為六種豬隻常見的混合性病毒感染，患豬臨床上呈現呼吸道症狀或繁殖障礙，導致疾病診斷的困難與經濟損失。即時聚合酶鏈鎖反應(real-time PCR)的分子診斷技術是藉由調整PCR反應中聚合酶、引子及探針的濃度及運用電腦軟體分析結果，同步判定檢體中是否有病原的存在（定性）及其濃度（定量）；且可在不同探針上標定不同波長的螢光物質，進一步建立於單一樣本的同一PCR反應管中同時偵測多重病原的通用反應條件。本研究利用ClontechR (Clontech laboratories, Inc., USA)所生產的試劑於MastercyclerR ep realplex (Eppendorf, Germany)建立上述六種病毒之多種偵測技術：SYBR Green real-time (RT-)PCR，其中PCV2、PPV及TTV1的偵測敏感度分別為2.27 ×101~10、1.76 × 101~10及4.47 × 100~9 copies/μL，PRRSV及CSFV的最低偵測敏感度則皆為101 TCID50/mL，並可進一步做為multiplex parallel SYBR Green real-time (RT-)PCR的使用；singleplex TaqMan real-time (RT-)PCR，PCV2、PPV及TTV1可進行定量的敏感度分別為2.2 ×101、1.57 × 101及4.47 × 100 copies/μL；PRRSV及CSFV的最低敏感度則分別為100 及101 TCID50/mL，而SIV相對於conventional RT-PCR也有較佳的敏感性，且自採樣檢體的萃取至偵測病毒感染，可在3~6小時內完成。另外，我們亦藉由調整引子及探針的濃度最佳化反應試劑的條件，嘗試建立duplex TaqMan real-time (RT-)PCR以便同步偵測PCV2與PPV或PRRSV與SIV；且若不經最佳化調整，在特定比例的濃度範圍以上，也可進行triplex TaqMan real-time (RT-)PCR來同步偵測各管中三種DNA或RNA病毒。以上技術的建立，可更快速及敏感的進行區別診斷，有助於豬隻疾病的防疫與監控工作。 | zh_TW |
| dc.description.abstract | Porcine circovirus (PCV2)、porcine reproductive and respiratory syndrome virus (PRRSV)、swine influenza virus (SIV)、Torque teno virus (TTV)、porcine parvovirus (PPV) and classical swine fever virus (CSFV) are the major pathogens of combined infection pigs. The combined infection not only causes serious economic loss but also increases the diagnostic difficulty. Real-time polymerase chain reaction (real-time PCR) is a powerful molecular technology for pathogen detection and quantification via a mathematical and computerizable way. The further development of multiplex real-time PCR can simultaneously analyze several targets in a single reaction, which also reduced sample volume, testing time, and cost. In the present study, we established the SYBR Green real-time (RT-)PCR for each of the above mentioned six important swine viral pathogens. The detection limit of the DNA/RNA assays are 22.7 copies/μL for PCV2, 17.6 copies/μl for PPV, 4.47 copies/μl for TTV1 and 101 TCID50/mL for PRRSV and CSFV. Besides, we also build singleplex TaqMan real-time (RT-)PCR. The detection limit of the DNA/RNA assays are 22 copies/μL for PCV2、15.7 copies/μl for PPV, 4.47 copies/μl for TTV1, 100 TCID50/mL for PRRSV, and 101 TCID50/mL for CSFV. Furthermore, the conditions of duplex TaqMan real-time (RT-)PCR for PCV2/PPV and PRRSV/SIV by decreasing concentrations of probe-primers and the detection limits of triplex TaqMan real-time (RT-)PCR for PCV2/PPV/TTV1 and PRRSV/SIV/CSF without optimization were both evaluated. These assays established may provide a more rapid and sensitive methodologies for swine disease diagnosis in the field. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T05:42:18Z (GMT). No. of bitstreams: 1 ntu-99-R97629004-1.pdf: 3924586 bytes, checksum: e1eb2d5912a632917f9e03dc63c94d03 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 口試委員會審定書…………………………………………………….……………….ii
致謝……………………………………………………………………………………..iii 目錄………………………………………………………………………...…………...v 圖次…………………………………………………………………………...……….iv 表次……………………………………………………………………………..…….xi 附錄………………………………………………………………………………........xiii 中文摘要…………………………………………………………………………...….xiv 英文摘要………………………………………………………………………….…...xv 第一章 序言………………………………………………………………………… 01 第二章 文獻回顧…………………………………………………………………… 03 第一節 常見豬隻呼吸道及繁殖障礙病毒性疾病………………………… 03 2.1-1 DNA/RNA 病毒簡介………………………………………………… 03 2-1-1-1. 第二型豬環狀病毒………………………………………… 03 2-1-1-2. 豬小病毒…………………………………………………… 04 2-1-1-3. 豬纖鍊病毒(Torque teno virus) …………………………… 05 2-1-1-4. 豬生殖與呼吸道綜合症病毒……………………………… 06 2-1-1-5. 豬流行性感冒病毒………………………………………… 07 2-1-1-6. 古典豬瘟病毒……………………………………………… 08 2.1-2 兩種或多種微生物的混合性感染…………………………………… 08 2-1-2-1. 豬隻離乳後多系統消耗性症候群………………………… 08 2-1-2-2. 豬增生性壞死性肺炎……………………………………… 09 2-1-2-3. 豬皮膚炎腎病症候群……………………………………… 10 2-1-2-4. 豬呼吸道疾病複合症……………………………………… 10 第二節 單一及多重即時聚合酶鍊鎖反應………………………………… 11 2-2-1. 即時聚合酶鍊鎖反應之原理………………………………………… 12 2-2-2. 多重即時聚合酶鍊鎖反應之限制及調整…………………………… 13 第三章 材料與方法………………………………………………………………… 15 第一節 實驗設計…………………………………………………………… 15 第二節 實驗材料…………………………………………………………… 16 3-2-1. DNA及RNA標準病毒之取得……………………………………… 16 3-2-2. 使用儀器及各商品化即時聚合酶鍊鎖反應試劑…………………… 16 3-2-2-1 Real-time 儀器及反應管…………..……………………… 16 3-2-2-2 商品化 SYBR Green 試劑………..……………………… 16 3-2-2-3 TaqMan primer/probe 試劑………..……………………… 17 3-2-3. 臨床血清樣本………..…………..……………………..……..……… 17 第三節 實驗方法………..……………..……………………..……..……… 17 3-3-1. DNA及RNA病毒之核酸萃取 ..……………………..……..……… 18 3-3-2. Real-time PCR之試劑選擇……..……………………..……..……… 18 3-3-3. 引子及探針之製備..……………..……………………..……..……… 18 3-3-4. 標準質體DNA製備……………..……………………..……….…… 19 3-3-4-1. 質體DNA培養及萃取 ……………………..……….…… 19 3-3-4-2. 限制酶切割酵素反應……………………..…………..…… 20 3-3-5. 利用 SYBR Green 進行單一及平行即時聚合酶鍊鎖反應....…..… 21 3-3-6. 利用 Taqman probe 進行單一即時聚合酶鍊鎖反應.……...…...……22 3-3-6-1. 引子及探針濃度調整.……………..…..….…..…...…..……23 3-3-6-2. 敏感性測試.…………….. ..….…....…..….…..…..…..……23 3-3-7. 利用 Taqman probe 進行多重即時聚合酶鍊鎖反應…..…...…..……24 3-3-7-1. 引子及探針濃度調整….....….…....…..….…..…...…...……24 3-3-7-2. 不經引子極探針濃度調整..….…....…..….…..…...…..……25 3-3-8. 數據分析….. .…..…...…...…....…..…....…...…....….…..…...…..……25 3-3-8-1. 標準曲線.....…...…....…..…....…...…....….…..…...…..……25 3-3-8-2. 四參數之雙彎曲函數(Four-parametric Sigmoid Function) ..25 3-3-9. 一般及反轉錄聚合酶鍊鎖反應(Conventional PCR & RT-PCR) ….…26 3-3-10. 統計分析………………………………………………………………..27 第四章 結果...…...…....…..…… .…..……..……..…..…...…....….…..…...…..……29 第一節 即時聚合酶連鎖反應試劑之選擇結果.....…....….…..…...…..……29 第二節 單一病毒即時聚合酶連鎖反應之建立....….....….…..…...…..……29 4-2-1 第二型豬環狀病毒.....…....…..….. ...…....….....….....…..…...…..……29 4-2-2 豬小病毒.…..……....….....…....... ...…......….....….....…..…...…..……31 4-2-3 第一型豬纖鍊病毒(Torque teno virus type 1) .....….....…..…...………32 4-2-4 豬生殖與呼吸道綜合症病毒....... ...…......….....….....…..…...…..……35 4-2-5 豬流行性感冒病毒...….....…....... ...…......….....….....…..…...…..……36 4-2-6 古典豬瘟病毒……....….....…..............…......….....….....…..…...…..…38 第三節 多重即時聚合酶連鎖反應通用條件之建立.....….....…..…...…..…39 4-3-1 引子及探針濃度調整…………………….…………………………… 39 4-3-1-1. DNA病毒....….. ..……................…......….....….....…..…...…..…39 4-3-1-2. RNA病毒.............……................…......….....….....…..…...…..…42 4-3-2 不經引子及探針濃度調整………………….……………...…………. 43 4-3-2-1. DNA病毒....….. ..……................…......….....….....…..…...…..…43 4-3-2-2. RNA病毒.............……................…......….....….....…..…...…..…43 第四節 臨床樣本核酸之檢測.. …................…......….....….....…..…...….…44 第五章 討論...…...…....…..…… .…..……..……..…..…...…....….…..…...…..……47 第六章 參考文獻..…....…..…… .…..……..……..…..…...…....….…..…...…..……56 | |
| dc.language.iso | zh-TW | |
| dc.subject | 豬隻呼吸道病毒性疾病 | zh_TW |
| dc.subject | 鏈鎖反應 | zh_TW |
| dc.subject | 同步偵測 | zh_TW |
| dc.subject | 平行偵測 | zh_TW |
| dc.subject | 多重病原診斷 | zh_TW |
| dc.subject | SYBR Green即時聚合酶 | zh_TW |
| dc.subject | 即時聚合酶 | zh_TW |
| dc.subject | 鏈鎖反應 | zh_TW |
| dc.subject | TaqMan即時聚合酶 | zh_TW |
| dc.subject | 鏈鎖反應 | zh_TW |
| dc.subject | Multiplex | en |
| dc.subject | Swine viral disease diagnosis | en |
| dc.subject | SYBR Green real-time (RT-)PCR | en |
| dc.subject | TaqMan real-time (RT-)PCR | en |
| dc.subject | Parallel | en |
| dc.title | 建立偵測常見豬隻呼吸道病毒性疾病即時聚合酶鍊鎖反應技術 | zh_TW |
| dc.title | Establishment of Real-time (RT-)PCR for Common Swine Respiratory Viral Disease Detection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 鄭謙仁,蕭世烜 | |
| dc.contributor.oralexamcommittee | 劉涓,張志成 | |
| dc.subject.keyword | 即時聚合酶,鏈鎖反應,豬隻呼吸道病毒性疾病,SYBR Green即時聚合酶,鏈鎖反應,TaqMan即時聚合酶,鏈鎖反應,同步偵測,平行偵測,多重病原診斷, | zh_TW |
| dc.subject.keyword | Swine viral disease diagnosis,SYBR Green real-time (RT-)PCR,TaqMan real-time (RT-)PCR,Multiplex,Parallel, | en |
| dc.relation.page | 128 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-20 | |
| dc.contributor.author-college | 獸醫專業學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| 顯示於系所單位： | 獸醫學系 | |
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