電磁式整合型微流體平台運用反轉錄恆溫式圈環形核酸增幅法於病毒偵測

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研究生：	蔡宇軒 Tsai, Yu-Shiuan
論文名稱：	電磁式整合型微流體平台運用反轉錄恆溫式圈環形核酸增幅法於病毒偵測 Electromagnetically-driven Integrated Microfluidic Platform Using Reverse Transcription Loop-mediated Isothermal Amplification for Viral Detection
指導教授：	李國賓 Lee, Gwo-Bin
口試委員:	陳致真沈延盛
學位類別：	碩士 Master
系所名稱：	工學院 - 動力機械工程學系 Department of Power Mechanical Engineering
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	94
中文關鍵詞：	新型冠狀肺炎、微流體、反轉錄恆溫式圓環形核酸增幅法、電磁鐵、微幫浦、微混合器、病毒偵測、定點照護
外文關鍵詞：	COVID-19, microfluidics, RT-LAMP, electromagnets, micropumps, micromixers, viral detection, point-of-care
相關次數：	點閱：3 下載：0
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對於嚴重急性呼吸道症候群冠狀病毒二型(SARS-CoV-2)快速且準確的偵測是非常重要的，目前主要是利用即時反轉錄聚合酶連鎖反應對新型冠狀肺炎進行檢測，然而這整個的過程通常會耗費4小時。取而代之的反轉錄恆溫式圈環形核酸增幅法是一種更加靈敏以及專一的核酸增幅法，並且在30~60分鐘之內就可以完成反應。本研究提出一個新型電磁驅動整合型微流體系統運用恆溫式圈環形核酸增幅法以及反轉錄恆溫式圈環形核酸增幅法伴隨著ＲＮＡ的萃取，並可以在60分鐘內完成整套流程且運用在新型冠狀病毒的快速偵測。藉由反轉錄恆溫式圈環形核酸增幅法結合ＲＮＡ的萃取後加上熒光偵測，快速並自動化診斷SARS-CoV-2可以被實現並且偵測極限可以達到5 x 103 copies/reaction。可攜式微流體裝置未來有希望可以用在一些資源比較有限的區域。

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important nowadays. Real-time PCR was commonly used for coronavirus disease diagnosis. However, it takes about up to four hrs to finish the whole process of amplification. Alternatively, reverse transcription loop-mediated isothermal amplification (RT-LAMP) could be more sensitive and specific, which could be finished within a shorter period of time (about 30~60 mins). In this work, a new electromagnetically-driven integrated microfluidic system was reported to perform the entire procedure for LAMP and RT-LAMP within 60 mins for fast diagnosis of SARS-CoV-2, followed by RNA extraction. By means of fluorescent detection after RT-LAMP with RNA extraction, fast diagnosis of SARS-CoV-2 could be automated with a LOD as low as 5 x 103 copies/reaction. The portable microfluidic device may be promising in resource-limited countries in the near future.

Abstract…………………………………………………………..................I
摘要………………………………………………………………………....II
Acknowledgements………………………………………………………..III
Table of contents…………………………………………………………..IV
List of figures……………………………………………………………..VII
List of tables…………………………………………………………….XVII
Abbreviations and nomenclature…………………………………….XVIII
Chapter 1: Introduction……………………………………………………1
1.1    Coronavirus
1.2      Reverse transcription loop-mediated isothermal amplification
1.3      Diagnostic methods for viral detection
1.4      Point-of-care diagnostics
1.5          Fluorescent dyes for RT-LAMP
1.6   Application of microfluidic with RT-PCR and RT-LAMP for         detection of SARS-CoV-2
1.7      Motivation and novelty
Chapter 2: Materials and methods……………………………………….16
2.1      Experimental process
2.2      Design of microfluidic chip
2.3      Microfabrication process
2.4      Sample and reagent preparation
2.5      Experimental setup
2.5.1      Optical detection module
2.5.2      Electromagnetic control module
2.5.3      Temperature control module
Chapter 3: Results and discussion………………………………………..41
3.1      Characterization of temperature control module
3.2      Characterization of microfluidic chip
3.3     Sensitivity  tests  by using cDNA samples for LAMP on chip for three genes
3.4     Specificity  tests  by using cDNA samples for LAMP on chip for three genes
3.5.     Optimization of calcein solution concentration
3.6.     Sensitivity  tests  by using synthetic RNA samples for RT-LAMP with RNA extraction on chip for three  genes
3.7.    Sensitivity tests by using synthetic RNA samples for fluorescent detection after RT-LAMP by using an ELISA reader and an optical setup
3.8.     Sensitivity tests with inactive virus samples
3.9      Clinical samples tests by using extracted RNA from patients
Chapter 4: Conclusions and future perspectives………………………...83
References…………………………………………………………………86
Publication list……………………………………………………………..94

                                

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