臺灣博碩士論文加值系統

新型冠狀病毒(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)從2019年12月 起在武漢引發「嚴重特殊傳染性肺炎」 (Coronavirus Disease-2019, COVID-19)。截至2021年1月底，全球確診感染人數已突破1億人，並確定200萬人因此死亡。新型冠狀病毒是一種高傳染性病毒，其主要傳播途徑為飛沫傳染。目前，除了將感染的病人隔離外，並沒有能有效治療COVID-19的藥物。但是，由於感染COVID-19 的病人在早期大多呈現與一般感冒相似的症狀，並且新型冠狀病毒能在人體潛伏14天或以上，因此很難在一般理學檢查中被篩檢出來。對於SARS-CoV-2病毒核酸，定量反轉錄聚合酶連鎖反應(Quantitative reverse-transcription polymerase chain reaction, qRT-PCR)是目前的黃金標準檢測方法。 然而，這個方法在執行上需要特殊的精密儀器進行檢測分析，也需要專業訓練的醫檢人員操作。這些條件都限制了qRT-PCR的使用範圍及可篩檢的樣本數目。因此，我們的目標是建立一個能夠適用於定點照護(point of care)且快速簡單從病人中檢驗新型冠狀病毒的方法。我們透過反轉錄恆溫環型核酸擴增法(Reverse transcription loop-mediated isothermal amplification, RT-LAMP)，建立一個能在恆溫下反應將目標核酸快速增幅的方式。為了方便判讀結果，加入了酚紅酸鹼指示劑，使RT-LAMP的增幅結果可以簡單地以肉眼判斷其顏色變化。由於新型冠狀病毒的核蛋白(Nucleoprotein, N)基因在不同地區的新型冠狀病毒分離株中都呈現高度的保留性，因此我們針對該基因設計了RT-LAMP的引子。根據我們的實驗結果，確認我們的引子能專一且靈敏地篩檢新型冠狀病毒核酸，並且能在30分鐘內得到檢測結果。概括而論，我們建立了一個快速篩檢SARS-COV-2 病毒的方法，而檢驗結果僅需肉眼即可觀察，我們期望將這個具潛力的核酸檢測方法應用於定點照護檢驗(point-of-care testing, POCT)。

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a serious outbreak of coronavirus disease COVID-19 pandemic since Dec 2019. As of Jan 31, 2021, more than 100 million confirmed cases and 2 million deaths have been reported worldwide. SARS-CoV-2 is a highly contagious virus which can be spreaded by droplet transmission. Currently, there are no effective therapies or vaccines against COVID-19, and the only way to prevent transmission of SARS-CoV-2 is to quarantine infected patients. However, it is difficult to detect SARS-CoV-2 in the early infection stage since most of the patients display flu-like symptoms or even asymptomatic. Also, the incubation period of COVID-19 can be up to 14 days. Currently, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is the most common and gold standard detection method for SARS-CoV-2 RNA. However, this method has several limitations, such as specialized equipment and skilled technicians. These strict requirements may limit the number that can be detected per period of time. Our goal is to develop a quick and simple SARS-CoV-2 screening test which can be delivered at the point of care. We utilize the reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is based on an isothermal nucleic acid amplification method, to develop the test. The RT-LAMP results with colorimetric changes can be determined easily by nude eye. Due to SARS-CoV-2 nucleoprotein (N) gene is highly conserved among the isolates, we designed a set of primers which specific bind to the SARS-CoV-2 N genes. Our RT-LAMP showed high specificity and sensitivity in detecting SARS-CoV-2 but not other coronaviruses nor common human respiratory disease-causing viruses within 30 minutes. Overall, we developed a RT-LAMP-based SARS-CoV-2 detection method, which delivered fast amplification and easy readout, with the potential to be applied to the point of care.

Table of Contents

誌謝 i
中文摘要 ii
Abstract iv
Table of Contents vi
List of Figures ix
List of Tables xi
Chapter 1 ： Introduction 1
1.1 Overview of the principle of Loop-mediated Isothermal Amplification (LAMP) 1
1.2 Mechanism of Loop-mediated Isothermal Amplification (LAMP) 5
1.3 Introductions to SARS-CoV-2 7
1.4 Introduction clinical workflow from sample collection to the analysis 12
1.5 The urgent demand of an alternative nucleic acid tests at point of care during SARS-CoV-2 outbreak 13
1.6 The LAMP/RT-LAMP assay for point-of-care testing 17
1.7 Specific aim 21
Chapter 2 ： Materials and Methods 22
2.1 Materials: 22
2.1.1 Cells 22
2.1.2 Competent cells 22
2.1.3 Primer 22
2.1.4 Plasmid 22
2.1.5 Reagent 23
2.1.6 Commercial kit 23
2.2 Methods 24
2.2.1 RT-LAMP primer design 24
2.2.2 RT-LAMP assay 25
2.2.3 RNA extraction by TRIzol 26
2.2.4 RNA extraction by GeneaidTM viral nucleic acid extraction kit 27
2.2.5 PCR amplification and sub-cloning 28
2.2.6 In vitro transcription with T7 RNA polymerase 29
2.2.7 Two step qRT-PCR 30
2.2.8 One step qRT-PCR 31
Chapter 3 ： Results 32
3.1 LAMP primer design and validation 32
3.2 The range of amplification temperature of the RT-LAMP assay 35
3.3 Specificity of the SARS-CoV-2 N-13 primer set 36
3.4 Sensitivity evaluation of the RT-LAMP assay with N-13 primer by the synthetic DNA and RNA samples 38
3.5 Validation of RT-LAMP assay by RNA from SARS-CoV-2 cell culture samples 41
3.6 QCMD blind test of in-house RT-LAMP assay 42
3.7 Validation of internal control primers targeting host genes 44
3.8 Validation of the performance of RT-LAMP assay under the background of human RNA 45
Chapter 4 ： Discussion 46
4.1 Point of care test is critical for controlling the SARS-CoV-2 pandemic 46
4.2 Colorimetric RT-LAMP assay has shown high potentials in point-of-care test 46
4.3 Difficulties and limitation of RT-LAMP to be improved 48
4.3.1 Aerosol transmission contamination 48
4.3.2 The selection of pH indicator 49
4.3.3 Mutations accumulated in the SARS-CoV-2 genome over time may affect primer efficiency 49
Chapter 5 ： Reference 50
Appendix 87
 
List of Figures
Figure 1. LAMP amplification mechanism 59
Figure 2. Discontinuous transcription of SARS-CoV-2 subgenomic mRNA 61
Figure 3. Alignment of the N gene sequences of seven coronaviruses 62
Figure 4. Alignment of N gene from SARS-CoV-2 isolate 64
Figure 5. N-1, N-13 and N-17 primers target regions in the SARS-CoV-2 N gene 65
Figure 6. N-1 and N-13 primers could successfully amplify the N gene cDNA in the colorimetric LAMP assay. 66
Figure 7. N-13 primer set amplified the SARS-CoV-2 N gene at 57.5 -70°C 67
Figure 8. N-13 primer set could not amplify the SARS-CoV-2 N gene at 55°C 68
Figure 9. N-13 primer sets specifically amplified N gene of SARS-CoV-2 but not HCoV-229E nor HCoV-OC43 69
Figure 10. N-13 primer sets specifically amplified N gene of SARS-CoV-2 but not SARS-CoV nor MERS-CoV 70
Figure 11. The respiratory disease-causing viruses were not detected by N-13 primer 71
Figure 12. Sensitivity evaluation of the LAMP assay in detecting commercially available N gene cDNA 72
Figure 13. Two-step qPCR result of Figure 12 73
Figure 14. In vitro transcribed SARS-CoV-2 N gene RNA was validated by electrophoresis in agarose gel 74
Figure 15. RT-LAMP assay with N-primer set was able to detect down to 10-10 dilution of RNA per reaction 75
Figure 16. One-step qPCR result of Figure 15 76
Figure 17. RT-LAMP assay was able to detect down to 0.02 fg RNA per reaction 77
Figure 18. RT-LAMP assay with the N-13 primer set has a detection limit in 20 fg 78
Figure 19. RT-LAMP assay could detect sample whose Ct value is below 33 79
Figure 20. QCMD samples were detected by RT-LAMP 81
Figure 21. Validation of internal control primers targeting host genes 82
Figure 22. Validation of the performance of RT-LAMP assay under the background of human RNA 83

 
List of Tables
Table 1. Primers used in (RT-)LAMP 84
Table 2. Primers used in cloning 85
Table 3. Primers used in qRT-PCR 85
Table 4. The RT-LAMP and qPCR result of Figure 19 86

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