本研究運用微流體技術及環形核酸恆溫增幅法針對新冠病毒中三個基因做快速檢測。RdRp、E和N，為構成新冠病毒中的病毒膜蛋白中的三個特定基因。此三種特定基因可用於偵測患者中的新冠病毒。此微流體平台利用微型控制器作為流體控制以及溫度控制，並搭配即時偵測螢光模組以用來快速檢測新冠病毒基因。研究結果顯示其在基因放大的過程中，其反應區域之溫度可以被精準地調控在0.5°C以內，整體實驗並可以在90分鐘內完成。實驗結果顯示若使用合成之病毒RNA，不活化病毒和從臨床檢體萃取中的病毒RNA來測試，其拷貝數數量在5000個以上，該系統可成功放大以及檢測檢體中的三個特定基因。如果用病毒的互補DNA，其系統對RdRp基因的檢測極限則可以到100個拷貝數，而對於E和N基因的檢測極限數則可以到1000個拷貝數。這個新的環形核酸恆溫增幅法即時偵測系統可作為檢測新冠病毒的平台，並且此小型檢驗系統因為其微型化和可攜性，更加適合作為定點照護的新冠病毒檢測系統。

A new integrated microfluidic platform utilizing real-time loop-mediated isothermal amplification (LAMP) for detecting three viral genes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for coronavirus diseases 2019 (COVID-19) was reported in this study. Three specific genes, including RNA dependent RNA polymerase (RdRp), E, and N genes encoding for the membrane proteins of viruses which are highly expressed in SARS-CoV-2 infected patients, were molecularly diagnosed. A compact integrated microfluidic system combining a microfluidic chip, a microcontroller, a fluidic control module, and a temperature control module was developed for rapid detection of RdRp, E, and N genes in SARS-CoV-2. Experimental results showed that the temperature inside the reaction chamber could be accurately regulated with a variation less than 0.5°C. The entire on-chip process including RNA extraction, reverse transcription and LAMP could be carried out in 90 min. Moreover, the developed system could successfully amplify RdRp, E, and N genes with a limit of detection (LOD) as low as 5x10^3 copies/reaction for each gene by using synthesized RNA, inactive viruses, and RNA extracts from clinical samples. The LOD could be as low as 10^2 copies/reaction for RdRp gene while 10^3 copies/reaction for E and N genes by using complementary DNA. This compact microfluidic platform may serve as a useful tool for SARS-CoV-2 point-of-care applications.

Abstract i
中文摘要 iii
誌謝 iv
Table of contents vi
Nomenclature and abbreviations x
List of tables xiii
List of figures xiv
Chapter 1 Introduction 1
1-1 Severe acute respiratory syndrome coronavirus 2 1
1-2 Loop-mediated isothermal amplification 2
1-3 Real-time LAMP 3
1-4 Microfluidic systems 4
1-5 Point-of-care (POC) 5
1-6 Motivation and novelty 5
Chapter 2 Materials and Methods 9
2-1 Preparation of magnetic beads and RT-LAMP reagent 9
2-2 Experimental procedure 12
2-3 Microfluidic chip design and fabrication 15
2-4 Real-time RT-LAMP device 20
2-4.1 Optical detection module 20
2-4.2 Pneumatic control module and electromagnetic valve control module 24
2-4.3 Temperature control module 28
2-4.4 Casing of the device 29
2-5 Agarose gel electrophoresis 32
2-6 Real-time RT-LAMP quantification 32
Chapter 3 Results and discussions 36
3-1 Characterization of temperature control module 36
3-2 Characteriztion of the microfluic chip 37
3-3 Specificity tests using sythesized RNA samples 41
3-4 Sensitivity test of LAMP using cDNA samples 42
3-5 Sensitivity tests including RNA extraction and RT-LAMP by using cDNA and synthesized RNA samples 45
3-6 Real-time RT-LAMP tests by using synthesized RNA samples 48
3-7 Sensitivity tests by using inactive virus samples 54
3-7.1 Sensitivity tests including virus lysis, RNA extraction, and RT-LAMP by
using inactive virus samples 54
3-7.2 Real-time RT-LAMP tests by using inactive virus samples 56
3-8 Sensitivity tests by using RNA extract of clinical samples 60
3-8.1 RNA extraction, and RT-LAMP by using RNA extract of clinical samples from 4 different outbreak areas 60
3-8.2-1 Sensitivity tests including RNA extraction, and RT-LAMP by using RNA extract of clinical samples from Taiwan 62
3-8.2-2 Real-time RT-LAMP tests by using RNA extract of clinical samples from Taiwan …………………………………………………………………………..64
3-8.3 Sensitivity tests including RNA extraction, and RT-LAMP by using RNA extract of clinical samples from England 68
Chapter 4 Conclusions and future perspectives 70
4-1 Conclusions 70
4-2 Future perspectives 71
References 73
Publication list..............................................................................................................77

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