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系統識別號 U0026-1608201611235600
論文名稱(中文) 快速簡易辨識登革熱感染症之概念性研發
論文名稱(英文) Conceptual Development of a Simple Rapid Method to Discriminate Dengue Virus Infection
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 104
學期 2
出版年 105
研究生(中文) 張沛宸
研究生(英文) Pei-Chen Chang
學號 P86034187
學位類別 碩士
語文別 英文
論文頁數 69頁
口試委員 指導教授-張憲彰
口試委員-黃執中
口試委員-葉才明
口試委員-彭貴春
口試委員-陳芃婷
中文關鍵字 登革熱診斷  登革熱非結構性蛋白(NS1)  磁性微珠 
英文關鍵字 Dengue diagnosis  Non-structural protein 1 (NS1)  Magnetic beads 
學科別分類
中文摘要 登革熱感染是一個全球性的議題,及重要性不容小覷。全世界每年有將近一億人感染登革熱,且死亡率極高。登革熱疫情主要集中在熱帶及亞熱帶國家,藉由病媒蚊所傳染。由於典型的登革熱症狀極輕微,類似一般感冒症狀,因此許多人往往不知道自己感染登革熱而忽視此疾病。但是當病人第二次感染被登革熱感染時,身體會產生劇烈的免疫反應,使病人有嚴重的症狀且極為痛苦,更可怕的是死亡率高達20%。在感染登革熱的早期,體內會出現病毒,病毒釋出的核酸及一種特殊的登革熱非結構性蛋白NS1,這些生物標的物在感染初期就能夠被檢測到,因此可以提供早期診斷的依據。在發病一個星期後,體內會開始產生免疫反應並出現相對應的免疫球蛋白,因此檢測體內所產生的免疫球蛋白也是另一種合適的診斷方式。現行檢測登革熱感染的方法有很多,都有其優點及缺陷。檢測體內的病毒及核酸含量,雖然其靈敏度高,但相對的需要耗費大量時間及金錢。利用酵素免疫分析法(ELISA)檢測體內中的NS1及免疫球蛋白,具有快速且方便操作的優點,但是靈敏度低。另外,市面上現在有兩款快篩套組,皆能夠在三十分鐘內完成診斷程序,但其靈敏度極低,大約60%而已。本研究開發了一套快速檢測登革熱感染的方式,利用磁性微珠具有好控制性且能夠表面修飾的優點,透過檢測樣本中的NS1,能夠在三十分鐘內完成所有診斷程序,且檢測極限為40 ng/mL,符合臨床檢測最低極限值,靈敏度高達90%。此方法使用肉眼就能輕易辨別結果,不需使用另外的儀器,可謂簡單又快速的登革熱檢測方法。此外,本研究也開發了一套影像判讀系統,能夠準確判斷樣本中NS1濃度,用以辨認病患為典型登革熱感染或出血性登革熱感染,準確率達85%。早期確診並提供支持性治療,能大幅降低病患死亡率。
英文摘要 Dengue disease is a global issue, and its importance should not be underestimated. There are almost 100 million people infected with dengue fever every year, and the disease has a high mortality rate. Many methods have been proposed for detection of the dengue virus infection, but all of them have both advantages and drawbacks. Virus and nucleic acid detection are highly sensitive, but they are time-consuming and expensive. It is easy and quick to use ELISA to detect NS1 and immunoglobulin, but the sensitivity is low. In addition, there are two rapid screening kits on the market now that are able to complete diagnostic procedures in 30 minutes, but their sensitivity is very low (about 60%). In this study, a rapid method for detection of dengue virus infection was developed through the use of magnetic beads. This method has the advantage of better controllability and is capable of surface modification. Through detecting the NS1 in a sample, all diagnostic procedures can be completed within 30 minutes, with a sensitivity of as much as 90% and a limit of detection of 40 ng/mL, which is consistent with the clinical detection minimum limit. The naked eye can easily discern the results without the use of additional instrumentation. Therefore, it can be described as a simple and rapid method for the detection of dengue fever. In addition, an image interpretation system was developed that can be used to accurately determine the concentration of NS1 in a sample, allowing identification of patients infected with dengue fever (DF) or dengue haemorrhagic fever (DHF) with an accuracy rate of 85%.
論文目次 Abstract i
摘要 ii
誌謝 iii
List of Figure vii
List of Table x
Chapter 1 Introduction 1
1.1 Background 1
1.2 Dengue 3
1.2.1 Epidemiology 3
1.2.2 Clinical Symptoms 6
(1) Febrile Phase 8
(2) Critical Phase 8
(3) Recovery Phase 8
1.2.3 Diagnosis 9
(1) Virus Isolation 11
(2) Nucleic Acid Detection 12
(3) IgM/IgG Testing 12
(4) Antigen Detection 12
(5) Rapid Detection of Dengue Disease 13
1.3 Magnetic beads 14
1.3.1 Magnetic Separations in Biotechnology 15
(1) Cell Separation 16
(2) Virus Separation 16
(3) Protein Separation 16
(4) Nucleic Acid Separation 17
1.4 Point-of-care 18
1.4.1 Definition of Point-of-care 18
1.4.2 Point-of-care Diagnostic Testing 18
1.4.3 Point-of-care for Biomedical Applications 19
1.5 Motivation and Research Framework 21
Chapter 2 Materials and Methods 22
2.1 Reagents and Instruments 22
2.1.1 Chemical materials 22
2.1.2 Solution 23
2.1.3 Instruments 23
2.2 Pretreatment of 33D2 antibody-biotin 24
2.3 Pretreatment of 31B2 antibody-HRP 25
2.4 The Evaluation of Ideal Immobilization Condition 26
2.5 The Procedure of Functionalized Magnetic Bead Pretreatment 28
2.6 The Procedure of Detecting rNS1 in PBS 29
2.6.1 Functionalized Magnetic Beads were Reacted with rNS1 and Detection Antibody Separately 29
2.6.2 Functionalized Magnetic Beads were Reacted with rNS1 and Detection Antibody Together 31
2.7 The Experiment to Determine the rNS1 in Plasma 32
2.7.1 The Evaluation of the Ideal Plasma Dilution Fold 32
2.7.2 The Procedure for Detecting Different Concentrations of rNS1 in Plasma 34
2.7.3 The Evaluation of the Ideal Reaction Time 35
2.8 Photographic Equipment 37
Chapter 3 Results and Discussion 39
3.1 Preparation of Functionalized Magnetic Beads 39
3.1.1 Ideal Immobilization Condition 39
3.2 Determine Different Concentrations of rNS1 in PBS 42
3.2.1 Functionalized Magnetic Beads were Reacted with rNS1 and the Detection Antibody Separately 42
3.2.2 Functionalized Magnetic Beads Reacting with rNS1 and the Detection Antibody Together 45
3.3 Determine Different Concentrations of rNS1 in Plasma 48
3.3.1 Ideal Plasma Dilution Fold 48
3.3.2 Functionalized Magnetic Beads Reacting with rNS1 and Detection Antibody Together in Plasma 50
3.3.3 Shortening of the Reaction Time 52
3.4 Image Analysis 54
3.4.1 Image Recognition System 55
3.4.2 Determine the Specific Concentration of rNS1 57
Chapter 4 Conclusion and Future Prospect 60
Acknowledgment 62
Reference 63
Curriculum Vitae 67
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