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研究生:許堯智
研究生(外文):Xu, Yao-Zhi
論文名稱:可攜式即時定量聚合酶連鎖反應系統
論文名稱(外文):A Portable Real-time Quantitative Polymerase Chain Reaction System
指導教授:馬席彬
指導教授(外文):Ma, Hsi-Pin
口試委員:蔡佩芸李國賓黃柏鈞
口試委員(外文):Tsai, Pei-YunLee, Gwo-BinHuang, Po-Chiun
口試日期:2021-01-21
學位類別:碩士
校院名稱:國立清華大學
系所名稱:電機工程學系
學門:工程學門
學類:電資工程學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:71
中文關鍵詞:即時定量聚合酶連鎖反應系統
外文關鍵詞:Real-time Quantitative Polymerase Chain Reaction
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這一年因為新型冠狀病毒(COVID-19)肺炎盛行導致全球人民動盪不安,早期偵測及診斷是遏止傳染疾病擴散的關鍵因素。因此許多研究學者投身於開發既 正確又快速的偵測工具。在所有偵測工具中,分子診斷已被廣泛的應用於感染病 偵測,因為具有高靈敏性和專一性。然而,市面上 real-time PCR 非常昂貴且不可攜帶,故只有在大型醫院才會有該設備,因此我們希望能做出一個可攜式且更快速,不僅成本更低,更能達到精準的診斷。
我們經由分離及純化後得到的核酸序列,有了這些資料後最快速的方法就是用即時定量聚合酶連鎖(Quantitative real-time PCR)反應偵測病毒核酸的存在,目前該系統是針對卵巢癌做檢測,但我們系統也可以依照各檢體需求不同,去調整系統參數進行更多疾病的檢測。
在這個系統中,我們將提供更輕易攜帶的尺寸(外殼長為30公分、寬為45公分、高為30公分)、並提供更快的檢驗速度(一次熱循環為 110.8 秒),相對於 市售儀器用更低的成本去完成一台即時定量連鎖聚合酶反應系統(Quantitative real-time PCR System)。
該系統主要分成三個子系統去進行控制,分別為溫度控制系統,雷射系統, 光電倍增管系統,經由這三個系統的組合可以形成一個即時定量連鎖聚合酶反應系統。溫度控制系統是將檢體進行複製,雷射系統是將完成複製後的檢體進行激發,光電倍增管系統是檢測完成激發檢體中的螢光量,由得到螢光量可以推算初原始濃度,並由此濃度去判斷是否有該疾病。
由於系統是自動的,使用者只要將微流道晶片放入並且按下開啟電源即可等待結果,而我們的溫度控制可以達到每秒升溫2.23度和降溫1.25度,並可以穩定的控制在正負1度,一次熱循環僅需 110.8 秒,而一小時內即可完成30次的熱循環。
In the past year, the prevalence of the deadly disease COVID-19 pneumonia has caused great anxiety among people around the world. Therefore, early detection and diagnosis is a key factor in curbing the spread of infectious diseases. At present, many researchers have focused on developing correct and fast detection tools. Among all detection tools, molecular diagnosis has been widely used in the detection of infectious diseases because of its high sensitivity and specificity. Nevertheless, the real-time polymerase chain reaction (PCR) that can be sold is very expensive and not portable, so this equipment is only available in large hospitals. In view of this situation, the team is eager to make a portable and faster, meanwhile product that is lower in cost and can achieve more accurate diagnosis.
After isolation and purification, our team obtained the nucleic acid sequence. With this information, the fastest method is to use quantitative real-time PCR to detect the presence of viral nucleic acid. As of today, the system previously only tested for ovarian cancer. However, the system of this study can also adjust system parameters to detect more diverse diseases according to the different needs of each sample. For this system, our team will provide a size that is easier to carry (case length is 30 cm, width is 45 cm, height is 30 cm) and faster inspection speed (a thermal cycle is 110.8 seconds). Compared with commercially available instruments, the product designed in this research can complete a quantitative real-time PCR system at a lower cost.
The system is mainly divided into three subsystems to perform control, namely tempera- ture control system, laser system, and photomultiplier tube system. The combination of these three systems forms a quantitative real-time PCR system. The purpose of the temperature control system is to copy the specimen, the laser system is to excite the copied specimen, and finally the photomultiplier tube system is responsible for detecting the amount of fluorescence in the specimen that has been excited. In the end, the initial original concentration can be es- timated by the amount of fluorescence obtained, and the concentration can further determine whether the patient has the disease.
This system is automatic. Users only need to put the micro-channel chip in and press to turn on the power and wait for the test result. The temperature control of the system can reach 2.23◦C and 1.25◦C per second, and can be stably controlled at plus or minus 1◦C. In other words, a thermal cycle only takes 110.8 seconds, and 30 thermal cycles can be completed in one hour.
Contents
Abstract i
1 Introduction 1
1.1 Background................................... 1
1.1.1 PolymeraseChainReaction ...................... 1
1.1.2 PCRSystemClassification....................... 4
1.1.3 Real-TimePolymeraseChainReaction ................ 5
1.1.4 Characteristics of Quantitative Real Time Polymerase Chain Reaction
andComparisonwithPCR....................... 8
1.1.5 ThresholdCycle ............................ 10
1.1.6 ApplicationofPCR........................... 12
1.2 Motivation.................................... 13
1.3 MainContributions ............................... 13
1.4 Organization................................... 15
2 Literature Review 17
2.1 PCR Instrument Temperature Control System Based on Multimodal Control . 17
2.2 Multimodal Control of PCR System Temperature System . . . . . . . . . . . 18
2.3 End-PointPCRSystem............................. 20
2.4 Comparison................................... 21
3 Real-Time Polymerase Chain Reaction 23
3.1 SystemOverview ................................ 23
3.2 MicrofluidicChip................................ 25
3.3 TemperatureControl .............................. 26
3.3.1 ThermoelectricCoolingModule.................... 27
3.3.2 Thermometer.............................. 29
3.3.3 SolidStateRelay............................ 30
3.3.4 PowerSupply.............................. 32
3.3.5 4-WayRelay .............................. 33
3.3.6 On-and-OffControlSystem ...................... 33
3.3.7 Proportional–Integral–Derivative Control System and Predictive Fil- terSystem ............................... 34
3.4 LaserModuleControl.............................. 36
3.5 Photomultiplier ................................. 37
Results and Discussion 45
4.1 PrototypeImplementation ........................... 45
4.2 SystemTesting ................................. 48
4.2.1 ExperimentalEnvironments ...................... 48
4.2.2 Temperature Experimental Data and Discussion . . . . . . . . . . . . 50
4.2.3 PCRResults .............................. 62
4.2.4 ComparisonandDiscussion ...................... 65
Conclusions and Future Works 67
5.1 Conclusions................................... 67 5.2 FutureWorks .................................. 68
5
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