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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 何佳安 | |
| dc.contributor.author | Jia-Yu Zheng | en |
| dc.contributor.author | 鄭嘉予 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:36:24Z | - |
| dc.date.available | 2022-09-12 | |
| dc.date.copyright | 2017-09-12 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77872 | - |
| dc.description.abstract | Human cytomegalovirus (HCMV) 人類巨細胞病毒又稱人類皰疹病毒第五型，是一種雙股DNA病毒，有非常強的傳染力。如果該病毒感染健康人時，通常不會觀察到明顯症狀，但若感染免疫不全的個體 (如:器官移植病人、AIDS病人和新生兒)，就可能會危及生命。另外，母體若於懷孕時期感染HCMV，病毒有很高的機率藉由胎盤、乳汁及唾液等途徑傳染給胎兒，造成新生兒出現呼吸困難、意識運動障礙、智力遲鈍、肝脾腫大、耳聾和中樞神經系統異常等嚴重的臨床症狀。目前診斷HCMV的方式之一為定量血液樣品中IgG及IgM的濃度；若兩者皆呈陽性反應，則過兩周後須再抽血定量一次。若血中IgG的量是前一次的四倍以上則表示已被感染HCMV。此方法雖為臨床常用的檢驗方法，但診斷結果無法即時得知。由於過去文獻曾指出，HCMV病毒帶有多個微小核酸片段分子(microRNAs)，並可在被感染的病人血液中被偵測出，因此設計一套表面電漿共振生醫感測系統，可以快速且準確檢測 HCMV感染，進而輔助醫生早期診斷，並適時給予適當藥物以預防母嬰垂直感染即為本研究的主要實驗目的。在這實驗中想利用表面電漿共振生醫感測器(surface plasmon resonance, SPR)來偵測血清中HCMV的microRNA，藉由磁珠抓住目標microRNA，再利用Poly A polymerase來將目標microRNA加上200~300 bases poly A tail ，利用Adenine對金有較強的吸附力，吸附在表面電漿共振系統偵測金表面上，造成表面電漿共振訊號改變來偵測是否有感染HCMV。目前實驗已經證明Poly A polymerase確實可以延長目標microRNA，而藉由表面電漿共振訊號圖，也可以證明經過延長的目標microRNA可以吸附在金表面上並且訊號有隨濃度的變化而提高。目前整個系統的偵測極限是在250pM。 | zh_TW |
| dc.description.abstract | Human cytomegalovirus (HCMV), known as human herpesvirus-5 (HHV-5), is a double strand DNA viruses. Vertical transmission of HCMV occurs frequently in newborns, then leads to congenital abnormalities. MicroRNAs (miRNAs) are short (18~23 nucleotide), non-coding nucleotides that play an important role in human diseases. The abundant expression of multiple microRNAs in blood that are encoded by HCMV, have been discovered after infection. In this study, we aimed to develop a surface plasmon resonance biosensing platform for the rapid detection of HCMV viral microRNA that can be used in the prediction of possible vertical transmission from mother to newborns. The utilization of the capture probe-modified magnetic nanoparticle enabled us to extract our target microRNA via biotin−streptavidin interaction. The function of later-added polymerase was used for elongation of our target microRNA. Finally the analytical measurement was performed on a surface plasmon resonance biosensing system to determine the amount of elongated nucleic acid sequences. The total assay time for our SPR sensing platform is less than 2 hours, and detection limit is calculated as 250 pM. This newly-developed sensing technology holds great promise in assisting physicians to facilitate treatment decision making, thus preventing timely congenital abnormalities in newborns. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:36:24Z (GMT). No. of bitstreams: 1 ntu-106-R04b22010-1.pdf: 2981262 bytes, checksum: 1e82cbbb0805214f25816178579114fc (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 第一章 緒論 1
1.1. 人類巨細胞病毒(Human Cytomegalovirus) 1 1.1.1. 人類巨細胞病毒傳播與病症 1 1.1.2. 人類巨細胞病毒現行診斷方法 2 1.2. 微小核糖核酸(microRNA) 3 1.2.1. microRNA在生物體扮演之功能 3 1.2.2. microRNA生合成及作用機制 3 1.2.3. microRNA與人類巨細胞病毒 4 1.2.4. microRNA之偵測方法 6 1.3. 核酸放大 8 1.3.1. 核酸放大技術 8 1.3.2. Poly A 聚合酶 9 1.4. 表面電漿共振生物感測器 10 1.4.1. 表面電漿共振生物感測器之特性 10 1.4.2. 表面電漿共振原理 11 1.4.3. 表面電漿共振分類 12 第二章 方法與材料 13 2.1. 材料 13 2.1.1. DNA及RNA序列 13 2.1.2. 實驗試劑與材料 14 2.1.3. 緩衝溶液 (Buffer) 16 2.2. 實驗儀器 17 2.3. 實驗方法 19 2.3.1. 聚丙烯醯胺膠體電泳(Polyacrylamide gel electrophoresis, PAGE) 19 2.3.2. 目標microRNA與探針雜合反應 19 2.3.3. Poly A polymerase延長作用之鑑定 20 2.3.4. Poly A polymerase延長作用以Real time PCR儀器偵測SYBR gold螢光 20 2.3.5. 磁珠-探針上進行microRNA 3端Poly A延長反應 21 2.3.6. Poly A polymerase 濃度最佳化 21 2.3.7. Poly A polymerase 反應時間最佳化 22 2.3.8. Poly A polymerase 反應最佳化以QPCR儀器偵測SYBR gold螢光 22 2.3.9. 磁珠-探針的製備 23 2.3.10. 表面電漿共振系統操作 24 2.3.11. Poly A 延長之產物吸附在金表面 26 2.3.12. 表面電漿共振系統應用於目標miR-UL22A-5p之分析 26 2.3.13. 選擇性(Selectivity)之測試 27 2.3.14. microRNA spike到老鼠血清中做測試 28 2.3.15. microRNA轉cDNA 29 2.3.16. microRNA 以QPCR kit測量 30 第三章 實驗結果與討論 31 3.1. 實驗設計 31 3.2. 磁珠製備與鑑定 33 3.2.1. 磁珠的非專一性吸附測試 33 3.2.2. 磁珠-探針的製備與鑑定 36 3.3. Poly A polymerase 延長反應 39 3.3.1. Poly A延長反應 39 3.3.2. 在磁珠-探針上進行microRNA 3端Poly A 延長反應 42 3.4. Poly A 延長反應之產物吸附在金表面 44 3.5. Poly A polymerase 反應條件最佳化 45 3.5.1. Poly A polymerase 濃度最佳化 45 3.5.2. Poly A polymerase 反應時間最佳化 47 3.5.3. 利用QPCR儀器最佳化poly A polymerase濃度與反應時間 49 3.6. 全反應測試 51 3.7. 選擇性(Selectivity)之測試 53 3.8. 血清測試 54 3.8.1. QPCR檢測Spike老鼠血清 54 第四章 結論 58 參考文獻 59 | |
| dc.language.iso | zh-TW | |
| dc.subject | 表面電漿共振生醫感測器 | zh_TW |
| dc.subject | 人類巨細胞病毒 | zh_TW |
| dc.subject | 微小核醣核酸 | zh_TW |
| dc.subject | Human cytomegalovirus | en |
| dc.subject | microRNA | en |
| dc.subject | poly A polymerase | en |
| dc.subject | surface plasmon resonance | en |
| dc.title | 發展早期診斷新生兒巨細胞病毒感染之表面電漿共振生醫感測器 | zh_TW |
| dc.title | Magnetic nanoparticle-assisted surface plasmon resonance biosensor for early diagnosis of human cytomegalovirus infection in newborns | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐士蘭,吳立真,陳平,楊家銘,鄭建中 | |
| dc.subject.keyword | 人類巨細胞病毒,微小核醣核酸,表面電漿共振生醫感測器, | zh_TW |
| dc.subject.keyword | Human cytomegalovirus,microRNA,surface plasmon resonance,poly A polymerase, | en |
| dc.relation.page | 64 | |
| dc.identifier.doi | 10.6342/NTU201703713 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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