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論文中文名稱:以金奈米粒子免疫色層分析法快速偵測沙門氏菌之研究 [以論文名稱查詢館藏系統]
論文英文名稱:Rapid detection of Salmonella using gold nanoparticle and
immunochromatography [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:化學工程研究所
畢業學年度:99
出版年度:100
中文姓名:陳柏豪
英文姓名:Po-Hao Chen
研究生學號:98738048
學位類別:碩士
語文別:中文
口試日期:2010-07-15
論文頁數:75
指導教授中文名:侯劭毅
口試委員中文名:王勝仕;黃光策;黃志宏
中文關鍵詞:免疫層析法沙門氏菌16S rDNA金奈米粒子
英文關鍵詞:Salmonella16SrDNAImmunochromatographyGold nanoparticles
論文中文摘要:近年來DNA分子檢測技術之應用方法發展迅速,臨床診斷上即常利用聚合酶鏈鎖反應(PCR)為重要輔助工具。PCR方法雖然準確,但耗時和耗材昂貴,尤其是及即時聚合酶鏈鎖反應(Real-time PCR)。基於上述考量,本實驗室開發了結合DNA分子與金奈米粒子免疫層析法,利用金奈米粒子與硫醇(thiol)之間有強大的共價鍵結力。先在DNA probe一端修飾硫醇基,使與金奈米鍵結,並運用此金奈米探針進行雜交( Hybrid ),捕捉位於沙門氏菌16S rDNA區域之目標DNA,再與標有 biotin之探針雜交後與卵蛋白(avidin)結合,再使用固定在硝化纖維膜上的抗生物素卵蛋白抗體(anti-avidin antibody)去捕捉卵蛋白,利用免疫層析法去偵測DNA。而此系統最大的優勢,在於不需要PCR放大,即可對一個菌落判斷是否為沙門氏菌,此方法也可明確區辨大腸桿菌和沙門氏菌。原本5fmol的偵測極限再經由銀還原訊號增強後對單股DNA可達5amol之檢測極限。並且可以肉眼直接判斷是否為沙門氏菌,也不需要光源激發和特殊儀器偵測,此研究不僅具快速、準確、操作簡便、低成本等優點,且兼顧高靈敏度與特異性,利於臨床使用與推廣。
論文英文摘要:In recent years, application of DNA molecular detection technology is developing rapidly, such as polymerase chain reaction is the important clinical diagnostic method. PCR method is accurate, but time-consuming, materials-consuming and other issues need to be improved. Therefore, our laboratory developed a immunochromatographic assay which uses DNA molecules, gold nanoparticles(GNPS) and antibodies.
We designed biotin-DNA and SH-DNA probes as capture and detection probes, each probe is complementary with Salmonella 16S rDNA. The biotin-DNA probe is associated with avidin and anti-avidin antibody on the nitrocellulose membrane, while the SH-DNA probe is connected to GNPS using covalent bond. With hybridization, immunochromatography methods are used to detect Salmonella.
The biggest advantage of this system is that does not require PCR amplification to determine whether a colony of Salmonella. This method can clearly distinguish E.coli between Salmonella. The detection limit of single-stranded DNA is 5 fmol. Furthermore, after silver enhancement, the detection limit is 5 amol. It is rapid, accurate, convenient, low cost and high sensitivity and specificity, which will help clinical use and promotion.
論文目次:摘 要 i
ABSTRACT ii
致 謝 iv
目 錄 v
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 前言 1
1.2 研究動機及目的 3
第二章 研究背景與原理介紹 4
2.1 沙門氏菌簡介 4
2.2 現行沙門氏菌的檢測方法 6
2.2.1 PCR 原理及檢測方法介紹 6
2.2.2 生化反應檢測法(Biochemical tests) 9
2.2.3 免疫分析法(Immunoassay) 11
2.3 金奈米粒子檢測技術介紹 13
2.3.1 膠體金結構 13
2.3.2 膠體金特性 14
2.3.3 膠體金光學性質 14
2.3.4 膠體金製備方法 15
2.3.5 金奈米的應用 15
2.4 免疫層析法(Immunochromatograpy) 19
第三章 實驗方法與步驟 25
3.1 實驗架構 25
3.2 設計探針 27
3.3 修飾硫醇DNA固定在金奈米粒子表面 28
3.4 硝化纖維膜固定anti-avidin antibody、avidin、avidin-biotin-DNAprobe的免疫層析法 29
3.4.1 膜上固定anti-avidin antibody偵測單股DNA 30
3.4.2 膜上固定avidin 偵測單股DNA 31
3.4.3 膜上固定avidin-biotin-DNA偵測單股DNA 32
3.5 銀還原訊號放大 33
3.6 DNA純化 34
3.7 DNA定量 34
3.8 免疫層吸法偵測雙股DNA 35
3.9 定量分析 36
3. 10 實驗藥品 37
3. 11 實驗儀器 38
第四章 結果與討論 40
4.1 偵測單股DNA實驗的最佳化條件 40
4.1.1 不同pH值狀態下的影響 40
4.1.2 不同鹽濃度狀態下的影響 41
4.1.3 以BSA做為Au-SH- DNA的封閉試劑 42
4.1.4 雜交時間以及avidin和biotin反應時間的影響 45
4.1.5 Au-SH-DNA的濃度對膜上訊號強度的影響 47
4.1.6 以最佳化條件進行免疫層吸法偵測單股DNA 49
4.2 膜上固定avidin和avidin-biotin-DNA probe偵測單股DNA 52
4.2.1 膜上固定avidin偵測單股DNA 52
4.2.2 膜上固定avidin-biotin-DNA probe偵測單股DNA 53
4.3 免疫層析法偵測沙門氏菌單一菌落culture sample 55
第五章 結論 61
參考文獻 62
附 錄 66
論文參考文獻:1. Bendayan, M. (2001). "Tech.Sight. Worth its weight in gold." Science 291(5507): 1363-1365.
2. Biagini, R. E., D. L. Sammons, et al. (2004). "Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins." Clinical and Diagnostic Laboratory Immunology 11(1): 50-55.
3. Bla kova, M., M. Koets, et al. (2009). "Development of a nucleic acid lateral flow immunoassay for simultaneous detection of Listeria spp. and Listeriamonocytogenes in food." European Food Research and Technology 229(6): 867-874.
4. Chiu, T. H., T. R. Chen, et al. (2005). "Sequencing of an internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. in food." International Journal of Food Microbiology 97(3): 259-265.
5. Clark, M. A. and E. L. Barrett (1987). "The phs gene and hydrogen sulfide production by salmonella typhimurium." Journal of bacteriology 169(6): 2391-2397.
6. Connelly, J. T., S. R. Nugen, et al. (2008). "Human pathogenic Cryptosporidium species bioanalytical detection method with single oocyst detection capability." Analytical and Bioanalytical Chemistry 391(2): 487-495.
7. Cudjoe, K. S., T. Hagtvedt, et al. (1995). "Immunomagnetic separation of Salmonella from foods and their detection using immunomagnetic particle (IMP)-ELISA." International Journal of Food Microbiology 27(1): 11-25.
8. D'Aoust, J. Y. (1991). "Pathogenicity of foodborne Salmonella." International Journal of Food Microbiology 12(1): 17-40.
9. Daniel, M. C. and D. Astruc (2004). "Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology." Chemical Reviews 104(1): 293-346.
10. Doyle, M. P. (1989). Foodborne bacterial pathogens, CRC.
11. Doyle, M. P., L. R. Beuchat, et al. (1997). Food microbiology: fundamentals and frontiers, ASM Press Washington, DC.
12. Edwards, K. A. and A. J. Baeumner (2006). "Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization." Analytical and Bioanalytical Chemistry 386(5): 1335-1343.
13. Euzéby, J. P. (1999). "Revised Salmonella nomenclature: Designation of Salmonella enterica Le Minor and Popoff 1987 sp. nov., nom. rev. as the neotype species of the genus Salmonella Lignieres 1900, rejection of the name Salmonella choleraesuis Weldin 1927, and conservation of the name Salmonella typh Warren and Scott 1930." International Journal of Systematic Bacteriology 49(2): 927-930.
14. Fisher, M., Y. Atiya Nasagi, et al. (2009). "A combined immunomagnetic separation and lateral flow method for a sensitive on site detection of Bacillus anthracis spores¡Vassessment in water and dairy products." Letters in applied microbiology 48(4): 413-418.
15. Gaillot, O., P. Di Camillo, et al. (1999). "Comparison of CHROMagar salmonella medium and Hektoen enteric agar for isolation of salmonellae from stool samples." Journal of clinical microbiology 37(3): 762-765.
16. Goo, V. Y. L., G. Q. L. Ching, et al. (1973). "Comparison of brilliant green agar and hektoen enteric agar media in the isolation of salmonellae from food products." Journal of Applied Microbiology 26(3): 288-292.
17. He, Y., S. Zhang, et al. (2011). "Ultrasensitive nucleic acid biosensor based on enzyme-gold nanoparticle dual label and lateral flow strip biosensor." Biosensors and Bioelectronics 26(5): 2018-2024.
18. Ho, J. A., S. C. Zeng, et al. (2008). "Liposome-based immunostrip for the rapid detection of Salmonella." Analytical and Bioanalytical Chemistry 391(2): 479-485.
19. Holmes, B., W. R. Willcox, et al. (1978). "Identification of Enterobacteriaceae by the API 20E system." Journal of Clinical Pathology 31(1): 22-30.
20. Jain, K. K. (2005). "Nanotechnology in clinical laboratory diagnostics." Clinica Chimica Acta 358(1-2): 37-54.
21. Joyanes, P., M. D. C. Conejo, et al. (2001). "Evaluation of the VITEK 2 system for the identification and susceptibility testing of three species of nonfermenting gram-negative rods frequently isolated from clinical samples." Journal of clinical microbiology 39(9): 3247-3253.
22. Kalogianni, D. P., L. M. Boutsika, et al. (2011). "Carbon nano-strings as reporters in lateral flow devices for DNA sensing by hybridization." Analytical and Bioanalytical Chemistry 400(4): 1145-1152.
23. Karami, A., R. Ranjbar, et al. (2007). "Rapid detection of different serovares of Salmonella entrica by multiplex PCR." Iranian Journal of Public Health 36(2): 38-42.
24. Katz, E. and I. Willner (2004). "Integrated nanoparticle¡Vbiomolecule hybrid systems: synthesis, properties, and applications." Angewandte Chemie International Edition 43(45): 6042-6108.
25. Kumar, S., K. Balakrishna, et al. (2005). "Rapid detection of Salmonella typhi in foods by combination of immunomagnetic separation and polymerase chain reaction." World Journal of Microbiology and Biotechnology 21(5): 625-628.
26. Ling, T. K. W., P. C. Tam, et al. (2001). "Evaluation of VITEK 2 rapid identification and susceptibility testing system against gram-negative clinical isolates." Journal of clinical microbiology 39(8): 2964-2966.
27. Liu, L., C. Peng, et al. (2007). "Development and evaluation of a rapid lateral flow immunochromatographic strip assay for screening 19 nortestosterone." Biomedical chromatography 21(8): 861-866.
28. Malorny, B., E. Paccassoni, et al. (2004). "Diagnostic real-time PCR for detection of Salmonella in food." Applied and environmental microbiology 70(12): 7046-7052.
29. Mao, X., Y. Ma, et al. (2009). "Disposable nucleic acid biosensors based on gold nanoparticle probes and lateral flow strip." Analytical chemistry 81(4): 1660-1668.
30. Mao, X., H. Xu, et al. (2009). "Molecular beacon-functionalized gold nanoparticles as probes in dry-reagent strip biosensor for DNA analysis." Chem. Commun.(21): 3065-3067.
31. Mirkin, C. A., R. L. Letsinger, et al. (1996). "A DNA-based method for rationally assembling nanoparticles into macroscopic materials." Nature 382(6592): 607-609.
32. Murray, P. R., W. L. Drew, et al. (1990). Medical microbiology, Wolfe Medical Publications Ltd.
33. Ngom, B., Y. Guo, et al. (2010). "Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: A review." Analytical and Bioanalytical Chemistry 397(3): 1113-1135.
34. Pan, T. M., T. K. Wang, et al. (1997). "Food-borne disease outbreaks due to bacteria in Taiwan, 1986 to 1995." Journal of clinical microbiology 35(5): 1260-1262.
35. Penn, S. G., L. He, et al. (2003). "Nanoparticles for bioanalysis." Current Opinion in Chemical Biology 7(5): 609-615.
36. Prescott, L., J. Harley, et al. (2002). "Microbiology, 5th." McGraw Hil1.
37. Rosi, N. L. and C. A. Mirkin (2005). "Nanostructures in biodiagnostics." Chemical Reviews 105(4): 1547-1562.
38. Ruiz, J., M. L. Núñez, et al. (1996). "Comparison of five plating media for isolation of Salmonella species from human stools." Journal of clinical microbiology 34(3): 686-688.
39. Ryan, K. J., C. G. Ray, et al. (2004). Sherris medical microbiology: an introduction to infectious diseases, McGraw-Hill Medical.
40. Salehi, T. Z., M. Mahzounieh, et al. (2010). "Detection of Salmonella serovars in zoo and pet reptiles, rabbits, and rodents in Iran by culture and PCR methods." Comparative Clinical Pathology 19(2): 199-202.
41. Sambrook, J. and D. W. Russell (2001). Molecular cloning: a laboratory manual, CSHL press.
42. Shenton, W., S. A. Davis, et al. (1999). "Directed Self Assembly of Nanoparticles into Macroscopic Materials Using Antibody¡VAntigen Recognition." Advanced Materials 11(6): 449-452.
43. Stewart, M. E., C. R. Anderton, et al. (2008). "Nanostructured plasmonic sensors." Chemical Reviews 108(2): 494-521.
44. Tanaka, R., T. Yuhi, et al. (2006). "A novel enhancement assay for immunochromatographic test strips using gold nanoparticles." Analytical and Bioanalytical Chemistry 385(8): 1414-1420.
45. Tataurov, A. V., Y. You, et al. (2008). "Predicting ultraviolet spectrum of single stranded and double stranded deoxyribonucleic acids." Biophysical chemistry 133(1-3): 66-70.
46. Thaxton, C. S., D. G. Georganopoulou, et al. (2006). "Gold nanoparticle probes for the detection of nucleic acid targets." Clinica Chimica Acta 363(1-2): 120-126.
47. Wallet, F., C. Loïez, et al. (2005). "Performances of VITEK 2 colorimetric cards for identification of gram-positive and gram-negative bacteria." Journal of clinical microbiology 43(9): 4402-4406.
48. Wang, S., C. Zhang, et al. (2005). "Development of colloidal gold-based flow-through and lateral-flow immunoassays for the rapid detection of the insecticide carbaryl." Analytica Chimica Acta 546(2): 161-166.
49. Wilson, R. (2008). "The use of gold nanoparticles in diagnostics and detection." Chem. Soc. Rev. 37(9): 2028-2045.
50. Yang, W., X. B. Li, et al. (2011). "A colloidal gold probe-based silver enhancement immunochromatographic assay for the rapid detection of abrin-a." Biosensors and Bioelectronics 26(8): 3710-3713.
51. Zhao, X., L. R. Hilliard, et al. (2004). "A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles." Proceedings of the National Academy of Sciences of the United States of America 101(42): 15027-15032.
52. 曾瑋佑(2008),利用奈米金粒子結合拓印法來偵測培養皿上的沙門氏菌,國立台北科技大學生物科技研究所碩士論文
53. 行政院衛生署食品藥物管理局(2011),康照洲局長
論文全文使用權限:同意授權於2016-03-28起公開