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
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 
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
參考文獻 1. Normile, D., Surprising new dengue virus throws a spanner in disease control efforts. Science, 2013. 342(6157): p. 415-415.
2. Murray, N.E., M.B. Quam, and A. Wilder-Smith, Epidemiology of dengue: past, present and future prospects. Journal of Clinical Epidemiology, 2013. 5: p. 299-309.
3. Dengue, W., dengue haemorrhagic fever. Factsheet No 117, revised May 2008. Geneva. World Health Organization, 2008. 2: p. 25-28.
4. Mahy, B.W. and M.H. Van Regenmortel, Desk encyclopedia of human and medical virology. 2010: Academic Press.
5. Simmons, C.P., et al., Dengue. New England Journal of Medicine, 2012. 366(15): p. 1423-1432.
6. Organization, W.H., et al., Dengue: guidelines for diagnosis, treatment, prevention and control. 2009: World Health Organization.
7. Chakravarti, A., R. Arora, and C. Luxemburger, Fifty years of dengue in India. Transactions of the Royal Society of Tropical Medicine and Hygiene, 2012. 106(5): p. 273-282.
8. Kakkar, M., Dengue fever is massively under-reported in India, hampering our response. British Medical Journal, 2012. 345.
9. Endy, T.P., et al., Determinants of inapparent and symptomatic dengue infection in a prospective study of primary school children in Kamphaeng Phet, Thailand. PLoS Neglected Tropical Diseases, 2011. 5(3): p. e975.
10. Gubler, D.J., Dengue and dengue hemorrhagic fever. Clinical Microbiology Reviews, 1998. 11(3): p. 480-496.
11. Bhatt, S., et al., The global distribution and burden of dengue. Nature, 2013. 496(7446): p. 504-7.
12. Reiter, P., Yellow fever and dengue: a threat to Europe. Eurosurveillance, 2010. 15(10): p. 19509.
13. Geneva, W., et al., Towards universal access: Scaling up priority HIV/AIDS interventions in the health sector: Progress report, 2009. 2009: World Health Organization.
14. Kumar, J., et al., Dengue haemorrhagic fever: An unusual cause of intracranial haemorrhage. Journal of Neurology, Neurosurgery & Psychiatry, 2007. 78(3): p. 253-253.
15. Ranjit, S. and N. Kissoon, Dengue hemorrhagic fever and shock syndromes*. Pediatric Critical Care Medicine, 2011. 12(1): p. 90-100.
16. Varatharaj, A., Encephalitis in the clinical spectrum of dengue infection. Neurology India, 2010. 58(4): p. 585.
17. Yip, W., Dengue haemorrhagic fever: Current approaches to management. Medical Progress October, 1980.
18. Rigau-Pérez, J.G., et al., Dengue and dengue haemorrhagic fever. The Lancet, 1998. 352(9132): p. 971-977.
19. Srikiatkhachorn, A., et al., Natural history of plasma leakage in dengue hemorrhagic fever: a serial ultrasonographic study. The Pediatric Infectious Disease Journal, 2007. 26(4): p. 283-290.
20. Soler, M., [Laboratory diagnosis to dengue virus infections]. Acta Científica Venezolana, 1997. 49: p. 25-32.
21. Innis, B., et al., An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. The American Journal of Tropical Medicine and Hygiene, 1989. 40(4): p. 418-427.
22. Paho, D., dengue hemorrhagic fever in the Americas: Guidelines for prevention and control, Scientific Publication No. 548. Pan American Health Organization, 1994.
23. Santé, O.m.d.l., Dengue Haemorrhagic Fever: Diagnosis, treatment, prevention and control. 1997: World Health Organization.
24. Falconar, A.K., E. de Plata, and C.M. Romero-Vivas, Altered enzyme-linked immunosorbent assay immunoglobulin M (IgM)/IgG optical density ratios can correctly classify all primary or secondary dengue virus infections 1 day after the onset of symptoms, when all of the viruses can be isolated. Clinical and Vaccine Immunology, 2006. 13(9): p. 1044-1051.
25. Pelegrino, J., Summary of dengue diagnostic methods. World Health Organization, Special Programme for Research and Training in Tropical Diseases, 2006.
26. Kuno, G., I. Gomez, and D. Gubler, An ELISA procedure for the diagnosis of dengue infections. Journal of Virological Methods, 1991. 33(1): p. 101-113.
27. Shu, P., et al., Comparison of a capture immunoglobulin M (IgM) and IgG ELISA and non-structural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections. Clinical and Diagnostic Laboratory Immunology, 2003. 10: p. 622-630.
28. Huhtamo, E., et al., Early diagnosis of dengue in travelers: comparison of a novel real-time RT-PCR, NS1 antigen detection and serology. Journal of Clinical Virology, 2010. 47(1): p. 49-53.
29. Tricou, V., et al., Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses. BMC Infectious Diseases, 2010. 10: p. 142.
30. Lima Mda, R., et al., Comparison of three commercially available dengue NS1 antigen capture assays for acute diagnosis of dengue in Brazil. PLoS Neglected Tropical Diseases, 2010. 4(7): p. e738.
31. Wang, S.M. and S.D. Sekaran, Early diagnosis of Dengue infection using a commercial Dengue Duo rapid test kit for the detection of NS1, IGM, and IGG. The American Journal of Tropical Medicine and Hygiene, 2010. 83(3): p. 690-5.
32. Thévenot, J., et al., Magnetic responsive polymer composite materials. Chemical Society Reviews, 2013. 42(17): p. 7099-7116.
33. Cho, S., et al. Diagnosis of Hepatitis C Virus (HCV) Through Affinity Chromatography using Photolabile Magnetic Beads Linked RNA Aptamer on a Chip. in Micro Total Analysis Systems 2002. 2002. Springer.
34. Borlido, L., et al., Magnetic separations in biotechnology. Biotechnology Advances, 2013. 31(8): p. 1374-85.
35. Melville, D., F. Paul, and S. Roath, High gradient magnetic separation of red cells from whole blood. IEEE Transactions on Magnetics, 1975. 11(6): p. 1701-1704.
36. Molday, R., S. Yen, and A. Rembaum, Application of magnetic microspheres in labelling and separation of cells. Nature, (1977): 437-438.
37. Chang, W.S., et al., Rapid detection of dengue virus in serum using magnetic separation and fluorescence detection. Analyst, 2008. 133(2): p. 233-40.
38. Gao, J., et al., Antibody affinity purification using metallic nickel particles. Journal of Chromatography B, 2012. 895: p. 89-93.
39. Borlido, L., et al., Stimuli‐Responsive magnetic nanoparticles for monoclonal antibody purification. Biotechnology Journal, 2013. 8(6): p. 709-717.
40. Zhang, Y., J. Bai, and J.Y. Ying, A stacking flow immunoassay for the detection of dengue-specific immunoglobulins in salivary fluid. Lab on a Chip, 2015. 15(6): p. 1465-1471.
41. Allen, G., Point-of-care diagnostic testing. Association of periOperative Registered Nurses Journal, 2008. 87(3): p. 641-643.
42. Zangheri, M., et al., A simple and compact smartphone accessory for quantitative chemiluminescence-based lateral flow immunoassay for salivary cortisol detection. Biosens Bioelectron, 2015. 64: p. 63-8.
43. Sperling, R.A. and W. Parak, Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles. Philosophical Transactions of the Royal Society of London A: Mathematical, Physical and Engineering Sciences, 2010. 368(1915): p. 1333-1383.
44. Theilacker, N., et al., Multiplexed protein analysis using encoded antibody-conjugated microbeads. Journal of The Royal Society Interface, 2011: p. rsif20100594.
45. Rusmini, F., Z. Zhong, and J. Feijen, Protein immobilization strategies for protein biochips. Biomacromolecules, 2007. 8(6): p. 1775-1789.
46. Hermanson, G.T., Bioconjugate techniques. 2013: Academic press.
47. Howarth, M., et al., A monovalent streptavidin with a single femtomolar biotin binding site. Nature Methods, 2006. 3(4): p. 267-273.
48. Libraty, D.H., et al., High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. Journal of Infectious Diseases, 2002. 186(8): p. 1165-1168.
49. Alcon, S., et al., Enzyme-linked immunosorbent assay specific to Dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. Journal of Clinical Microbiology, 2002. 40(2): p. 376-381.
50. Lin, S.W., et al., Dengue virus nonstructural protein NS1 binds to prothrombin/thrombin and inhibits prothrombin activation. J Infect, 2012. 64(3): p. 325-34.
  • 同意授權校內瀏覽/列印電子全文服務,於2018-08-04起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2018-08-04起公開。

  • 如您有疑問,請聯絡圖書館