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研究生:葉品寬
研究生(外文):Pin Kuan Yeh
論文名稱:利用光熱系統發展核酸定性分析平台進行快速細菌偵測
論文名稱(外文):Development of a qualitative nucleic acid analysis platform for rapid bacterial detection based on photothermal system
指導教授:吳旻憲
指導教授(外文):M. H. Wu
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物科技產業碩士學位學程
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:116
中文關鍵詞:細菌檢測恆溫環狀擴增法光熱奈米粒子結核菌
外文關鍵詞:bacterial detectionLAMPphoto-thermal nanoparticlesMycobacterium tuberculosis
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細菌檢測是傳染性疾病預防與治療之關鍵。在眾多檢測方法中,細菌培養檢測法因具有高靈敏度與專一性而被視為黃金標準法,然而此技術需要消耗大量的培養時間以進行後續的診斷與治療,對於傳染性疾病而言,傳染的擴大與致死率的提高往往是因為診斷的拖延。因此疾病早期診斷與治療迫切需要高靈敏度且快速的細菌檢測系統。核酸放大檢測法的出現提供了更快且靈敏的細菌檢測方式,其中恆溫環狀擴增法(loop-mediated isothermal amplification, LAMP)是種能夠複製大量核酸產物的恆溫反應,曾應用於許多臨床實驗以及食物安全檢測,但過去並沒有研究將快速、簡易的溫控系統與恆溫環狀擴增法做整合。
本研究以近紅外雷射驅動具備光熱特性之磁性奈米粒子並與恆溫環狀擴增法結合,其目的要建構能快速定性分析細菌之光熱整合型平台。本實驗以結核菌16s rRNA片段基因作為實施項目並評估所設計的引子在此系統下之反應執行效率。結果顯示,光熱恆溫環狀擴增系統可在18秒內將溫度上升到系統反應所需的65度,而此系統在反應時間30分鐘下之核酸偵測極限低於100個拷貝數,靈敏度高於傳統PCR儀系統(1000個拷貝數)。此外,結果顯示本系統10分鐘即可完成反應。綜合上述,本系統具備極大的潛力能夠開發成快速且簡易的定性細菌檢測平台,並在未來的臨床檢驗與即時檢測系統有相當大的應用性。
The bacterial detection is vital for the prevention and treatment of infectious bacterial diseases (e.g., Tuberculosis). Among the methods of bacterial detection, bacterial culture has been recognized as the gold standard for bacterial identification due to its high sensitivity and specificity. However, this technique takes a lot of time to perform the bacteria culture, thus postponing subsequent diagnosis and treatment. The delayed treatment leads to the spread of disease and raise the mortality of patients. Therefore, it is urgent to develop a rapid and sensitive detection system for early bacterial diagnosis and treatment. To address it, the emergence of Nucleic Acid Amplification Testing (NAAT) provides a faster and more sensitive method for bacterial detection, in which loop-mediated isothermal amplification (LAMP) has been regarded as an alternative PCR-based methodology which can achieve the high-efficiency amplification of target gene. LAMP technique has been widely used for bacterial detections in clinical laboratory and food safety testing. However, in past researches, none of them have been investigated to integrate a fast and simple temperature control system with the LAMP.
Hence, in this study, we combined the temperature control system consisting of near-IR triggered magnetic photo-thermal nanoparticles with the LAMP technique to establish an integrated platform, termed as photonic LAMP system, utilized to perform a qualitative analysis for rapid bacterial detection. To evaluate the nucleic acids amplification performances of photonic LAMP system, we designed the LAMP primer sets specified for 16S rRNA of Mycobacterium tuberculosis (MTB). The results showed that the photonic LAMP system was capable of rapidly heating the solution to 65°C within 18 seconds. In 30 minutes reaction of nucleic acids amplification, the limit of detection (LoD) of the photonic LAMP system (<102 copies) was lower than that of conventional PCR machine (103 copies). Furthermore, the nucleic acids amplification reaction in photonic LAMP system was able to be accomplished within 10 min. These results indicate that photonic LAMP system provides a rapid and simple platform for qualitative bacterial detection, which holds great potential for clinical diagnostics and point-of-care detection.
指導教授推薦書
口試委員審定書
誌謝...iii
中文摘要...iv
Abstract...vi
目錄...viii
圖目錄...xiii
表目錄...xix
第一章、 前言...1
第二章、 文獻回顧...3
2.1 細菌檢測...3
2.1.1 細菌檢測方法...3
2.1.2 結核病簡介...5
2.1.3 結核病之檢測...5
2.2 核酸檢測系統...7
2.2.1 聚合酶連鎖反應...7
2.2.2 聚合酶連鎖反應與微機電之整合系統...11
2.2.3 聚合酶連鎖反應微流體整合系統之溫度控制...12
2.3 恆溫環狀擴增法...16
2.3.1 技術簡介...16
2.3.2 恆溫環狀擴增法之檢驗方法...19
2.3.3 恆溫環狀擴增法之整合系統...23
2.4 奈米粒子...26
2.4.1 奈米粒子之光熱效應與表面電漿共振...26
2.4.2 奈米粒子的表面修飾...29
第三章、 研究動機與目的...31
第四章、 材料與方法...34
4.1 系統流程與架構...34
4.2 反應載具設計...37
4.3 實驗儀器架設...40
4.3.1 雷射硬體...40
4.3.2 輸出功率控制與檢測...41
4.4 光熱奈米粒子分析...41
4.4.1 紫外光/可見光/近紅外光光譜儀...42
4.4.2 光熱效應分析...42
4.5 LAMP反應檢測...43
4.5.1 LAMP試劑...44
4.5.1.1 引子設計與質體合成...44
4.5.1.2 螢光染劑...46
4.5.1.3 LAMP反應試劑...46
4.5.1.4 臨床檢體...46
4.5.2 凝膠電泳...47
4.5.3 螢光訊號檢測...48
4.5.4 螢光訊號分析方法...48
第五章、 結果與討論...50
5.1 市售奈米磁珠粒子...50
5.1.1 基礎材料測試...50
5.1.1.1 紫外光/可見光/近紅外光吸收光譜分析...50
5.1.1.2 奈米粒子光熱轉換參數測試...51
5.1.1.3 奈米粒子光熱轉換穩定度...53
5.1.1.4 懸浮性測試...55
5.1.2 LAMP反應...57
5.1.2.1 傳統PCR儀、photonic LAMP加熱系統之LAMP反應比較...57
5.1.2.2 驗證鏈親合素/生物素鍵結...60
5.1.2.3 以奈米粒子作為訊號載體並比較傳統PCR儀系統與Photonic LAMP系統之LAMP反應差異...62
5.1.2.4 LAMP反應時間之螢光訊號分析...66
5.2 光熱反應材料與螢光染劑選用...68
5.2.1 複合型磁性奈米高分子粒子...68
5.2.2 EvaGreen螢光染劑...70
第六章、 補充資料...71
6.1 複合型磁性奈米粒子...71
6.1.1 穿透式電子顯微鏡成像...71
6.1.2 紫外光/可見光/近紅外光吸收光譜分析...73
6.1.3 奈米粒子光熱轉換參數測試...75
6.1.4 奈米粒子與LAMP試劑的沉澱特性...77
6.1.5 驗證鏈親合素/生物素鍵結...80
6.2 LAMP反應與螢光分析...82
6.2.1 引子與奈米粒子預先鍵結之LAMP反應...82
6.2.2 EvaGreen螢光染劑測試...84
第七章、 結論...86
第八章、 未來展望...87
第九章、 參考文獻...88



圖目錄
圖 1 聚合酶連鎖反應主要流程包刮:變性(Denaturation)、引子的黏合(Annealing of primers)與引子的延長(Extension of primers)[10]...10
圖 2 (A)鉑鈦加熱系統示意圖[20]。 (B)熱空氣加熱法之裝置示意圖[21],以馬達將熱空氣通入系統,並以擋板切換控制熱空氣 (C)紅外線加熱法示意圖[23],以近紅外光源通過濾鏡後對焦於加熱反應區 (D)微波加熱法示意圖[25] (E)奈米粒子媒介光熱系統示意圖,以奈米金桿作為雷射接收載體,同時亦為系統溫度控制之熱源,可調整功率以提供聚合酶連鎖反應所需溫度[26]。...15
圖 3 LAMP反應流程示意圖(A) LAMP反應所需的兩種特殊引子內引子(FIP/BIP)、外引子(F3/B3)。 (B) FIP, BIP所合成的互補DNA被上游的F3, B3引子鏟起,因兩端有特殊互補序列,因此聚合過程中形成一個環狀結構。 (C)FIP, BIP引子在圈環處繼續聚合,使環狀結構DNA不斷延伸,且會有部分產物會重新回到起始放大循環[31]。...18
圖 4 (A) Ranjbar R.等人以觀察副產物焦磷酸鎂之白色沉澱以判斷LAMP反應之陰陽性[35]。 (B)Norihiro Tomita等人以鈣黃綠素受鎂離子誘導產生螢光的原理,透過365nm紫外光照射觀察的螢光訊號[31]。 (C) Goto等人透過羥基萘酚藍與鎂離子的交互作用觀察顏色差異,會由陰性組別呈現紫色,而陽性組別呈現淡藍色[38] (D) Safavieh等人提出掃描伏安法(LSV)測量核酸產物訊號之系統示意圖[39]。...22
圖 5 (A) Garrido-Maestu等人透過特殊修飾的金奈米粒子做為指示劑與LAMP試劑中的鎂離子作用,若反應為陽性溶液呈現紫色[51]。 (B) Chen, W.等人在微流道中加入針對四種不同菌的引子,以EvaGreen做為指示染劑,並透過手機與簡單光學元件整合成偵測系統[52]。 (C)透過微流體特殊設計以油相流體包裹LAMP反應樣品成水相微粒,並在流道設計微粒儲存槽方便加熱反應,觀察鈣黃綠素呈現的螢光訊號以判斷反應結果[55]。...25
圖 6 實驗流程示意圖,本實驗預期流程分為(1)細菌篩選純化、(2)細菌光熱裂解、(3)核酸放大、(4)核酸產物偵測、(5)螢光訊號分析、(6)系統模組化等六大部份。...36
圖 7 載具設計示意圖,利用軟體Solidwork繪製翻模模組,並利用微流道銑雕機以壓克力板為材料雕刻。...38
圖 8 (A)載具示意圖,以PDMS為基材,透過翻模形成流道,其中包含反應腔室、液體出入孔、感溫線孔,並將PDMS與蓋玻片接合組成。 (B)注入反應試劑後在明視野下的載具側視圖。(C)反應溶液以染劑染色後放在藍光觀測台中的情形。...39
圖 9 雷射硬體系統示意圖,其中包含驅動主機、雷射光源以及塑膠微管載台。...40
圖 10 市售磁性奈米粒子之紫外光/可見光/近紅外光吸收光譜。...50
圖 11 以市售磁性奈米粒子配製Blank以及濃度2000 ppm、4000 ppm、6000 ppm、8000 ppm、10000 ppm的LAMP溶液,以(A) 1 W、(B) 2 W的雷射功率照射加熱,並以溫度記錄器搭配感溫線記錄溫度趨勢。黃色區域為適合LAMP反應之溫度區間。...52
圖 12 以1 W功率雷射照射含10000 ppm奈米粒子之LAMP溶液,反覆升降溫並紀錄溫度趨勢。...54
圖 13 配製含有2000 ppm市售磁性奈米粒子之LAMP溶液,在室溫下20分鐘的沉澱狀況。...56
圖 14 傳統PCR儀、photonic LAMP加熱系統之LAMP反應比較。含有0~105起始核酸拷貝數的反應試劑,(A, 上排)以PCR儀執行(65°C 20 min, 80°C 5 min),反應後染色並在藍光觀測台下溶液的螢光呈現。(A, 下排)以凝膠電泳法呈現相對核酸大小分布的情形。(B)將帶有螢光訊號的樣品移至qPCR儀分析螢光訊號讀值(以0拷貝數測到的螢光讀值為設為一倍,其餘按倍數關係呈現)。(C, 上排)以雷射執行(65°C 20 min, 80°C 5 min),反應後染色並在藍光觀測台下溶液的螢光呈現。(C, 下排)以凝膠電泳法呈現相對核酸大小分布的情形。(D)最後將帶有螢光訊號的樣品移至qPCR儀分析螢光訊號讀值(以0拷貝數測到的螢光讀值為設為一倍,其餘按倍數關係呈現)。...59
圖 15 (A)LAMP反應為陰性,將含有反應後奈米粒子的溶液移至螢光顯微鏡下觀察,在20x10倍率視野下的明視野(上)與螢光視野(下)。(B)LAMP反應為陽性。...61
圖 16以奈米粒子作為訊號載體並比較傳統PCR儀系統與Photonic LAMP系統之LAMP反應差異(A)、(B)、(C)在PCR儀溫控系統下執行LAMP,(D)、(E)、(F) 以Photonic LAMP系統之雷射溫控執行LAMP,兩系統中同時配製了B(無核酸模板, Blank)、10、102 、103、105拷貝數核酸模板的LAMP反應溶液。(A)、(D)反應後染色並在藍光觀測台下所呈現情形,(B)、(E)將奈米粒子沖洗2次並移至即時連鎖聚合反應儀中以螢光分析系統螢光讀值,(C)、(F) 將上清液移至即時連鎖聚合反應儀中分析螢光讀值...65
圖 17以Photonic LAMP系統執行反應,雷射誘導加熱維持在65度並以不同反應時間參數執行,分別為0、10、20、30分鐘。反應結束後將奈米粒子以去離子水沖洗並重新懸浮,接著將奈米粒子溶液與上清液移至即時連鎖聚合反應儀中分析螢光讀值。...67
圖 18 (A)穿透式電子顯微鏡視野下的複合型磁性奈米粒子。(B)將此奈米粒子溶液以雷射照射並反覆升降溫後,在穿透式電子顯微鏡視野下之成像。...72
圖 19將複合型磁性奈米粒子與有修飾鏈親合素的複合型磁性奈米粒子,分別稀釋成200 μg/ml與20 μg/ml兩種不同濃度後以紫外光/可見光/近紅外光吸收光譜儀分析,S-,沒有修飾鏈親合素的複合型磁性奈米粒子,S+, 有修飾鏈親合素的複合型磁性奈米粒子。...74
圖 20以複合型磁性奈米粒子配製成特定濃度的LAMP溶液,以雷射照射加熱並以溫度記錄器搭配感溫線記錄不同雷射功率下的溫度趨勢。黃色區域為適合LAMP反應之溫度區間。...76
圖 21配製2000 ppm的複合型磁性奈米粒子之LAMP溶液,在室溫下20分鐘的沉澱狀況。...78
圖 22 (A)不同溶劑與示意圖編號中不同溶質相互混合反應,並觀察10分鐘內之沉澱現象,√: 有沉澱,X: 無沉澱,-: 未測試。(B)將複合型磁性奈米粒子與LAMP試劑中不同成分單獨混合,並放置10分鐘後之沉澱現象。...79
圖 23 (A)管1為未加核酸模板的陰性控制組,管2至管6為有加核酸模板的陽性控制組,染SYBR Safe後於藍光觀測台紀錄。(B)管1、2未加入粒子,管3加入1200 ppm未修飾鏈親合素之奈米粒子,管4至管6分別加入600、1200、1800 ppm修飾有鏈親合素的奈米粒子,並混勻在室溫下作用1小時。(C)以強力磁鐵將粒子收集於底部。(D)、(E)將管3上清液去除後以去離子水重新懸浮粒子,並製成玻片樣本後,在螢光顯微鏡20 X 10視野下觀察紀錄(D)明視野與(E)螢光視野。(F)、(G) 將管5上清液去除後以去離子水重新懸浮粒子,並製成玻片樣本後,在螢光顯微鏡20 X 10視野下觀察紀錄(F)明視野與(G)螢光視野。...81
圖 24使用PCR儀設定65度加熱LAMP反應試劑30分鐘,結束後以染劑SYBR Safe染色,將奈米粒子與上清液分別以即時連鎖聚合反應儀分析螢光讀值。-particle-primer為引子是自由狀態下執行LAMP反應。+particle-primer為引子FIP/BIP預先和奈米粒子鍵結才執行LAMP反應。...83
圖 25比較在LAMP反應前預先加入EvaGreen與LAMP反應後加入SYBR Safe,兩個染劑在藍光觀測台下的結果。-:未加入複合型磁性奈米粒子; +: LAMP試劑中預先加入複合型磁性奈米粒子;+*: LAMP反應結束後再加入複合型磁性奈米粒子混合; N: LAMP試劑中不包含核酸模板的陰性組; P: LAMP試劑中包含104拷貝數的核酸模板的陽性組。...85



表目錄
表 1 Mycobacterium tuberculosis 16S rRNA基因片段的六條LAMP引子序列設計。...45
表 2 Mycobacterium tuberculosis H37Ra菌株的16S rRNA基因中一段長度211bp的片段以及本實驗所設計引子之辨認位置。...45
表 3 記錄圖13每個光熱循環在關掉雷射前30秒的溫度平均,並計算每次循環之 變異係數。...54
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