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論文中文名稱:金奈米粒子免疫色層分析法偵測沙門氏菌之臨床試驗 [以論文名稱查詢館藏系統]
論文英文名稱:Detection of Salmonella in the clinical samples 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:生物科技研究所
畢業學年度:100
出版年度:101
中文姓名:曾鈺婷
英文姓名:Yu-Ting Tseng
研究生學號:99688009
學位類別:碩士
語文別:中文
口試日期:2012-07-27
論文頁數:61
指導教授中文名:侯劭毅
口試委員中文名:黃志宏;黃光策;王勝仕
中文關鍵詞:免疫層析法沙門氏菌金奈米粒子16S rDNA
英文關鍵詞:Salmonella16S rDNAImmunochromatographyGold nanoparticle
論文中文摘要:沙門氏菌在自然界中為一個相當重要的病原菌,大致上可以分成兩種類型,一種是屬於傷寒型的沙門氏菌感染,造成所謂的傷寒症狀;另一種為非傷寒沙門氏菌,感染會造成腸道炎。沙門氏菌的感染引發急性腸胃炎在台灣地區的夏天為常見的傳染性疾病。在這篇研究裡,我們利用奈米金粒子的視覺特性並且純化沙門氏菌的DNA及RNA,設計針對沙門氏菌在16S rDNA序列的專一探針,以免疫層析法的方式來偵測沙門氏菌,並且將這個方法應用在臨床病人檢體的分析上面,可以快速、方便的來做到沙門氏菌的偵測。在這個實驗中,我們總共收集了1278個病人的糞便檢體,並將這些檢體塗盤在Hektoen agar上面進行初步篩檢,其中有159糞便檢體長出黑色的菌落,以這159個糞便檢體進行進一步的研究分析,同時以vitek 2菌種分析做為比對,結果顯示有80個檢體確實為沙門氏菌感染,偵測的靈敏度也達到了100%,其中有75個檢體也確實為非沙門氏菌感染,偵測的特異性為94.93%。我們設計了這個快速、低成本的實驗,希望可以透過這個檢測方法,取代臨床上對糞便檢體耗時的生化測試。
論文英文摘要:Salmonella spp. are important bacterial pathogens in the worldwide. In this study, we use the visual characteristics of nano-gold particles and purified Salmonella DNA and RNA, the design of specific probes for Salmonella, analysis of the way to detect Salmonella, and the patients in the clinical application of this method analysis of samples above, we hope the study can quickly and easily to achieve the detection of Salmonella. On Hektoen agar, 159 stool samples containing black or crystalloid colonies were identified using the proposed method. Without any equipment, such as microscope, the red band corresponding Salmonella were observed. After immunochromatography test, 80 of the isolates were correctly identified as Salmonella to achieve 100% sensitivity. 75 samples were correctly identified as non-Salmonella spp., but 4 was incorrectly identified as Salmonella. The specificity is 94.93%. We designed this rapid, convenient, low cost and high sensitivity and specificity experiment, which will help clinical use and promotion.
論文目次:摘要 i
ABSTRACT ii
誌謝 iii
目錄 iv
表目錄 vii
圖目錄 viii
第一章 緒論 1
1.1 前言 1
1.2 研究動機及目的 4
第二章 研究背景及原理介紹 5
2.1 沙門氏菌簡介 5
2.1.1 沙門氏菌之特性 5
2.1.2 沙門氏菌之分類 6
2.1.3 沙門氏菌之血清型 6
2.2 金奈米粒子檢測技術介紹 7
2.2.1 膠體金結構 8
2.2.2 膠體金特性 8
2.2.3 膠體金光學性質 9
2.2.4 膠體金製備方法 9
2.2.5 金奈米的應用 10
2.2.6 金奈米與DNA的結合 11
2.2.7 金奈米與蛋白質的結合 13
2.3  沙門氏菌之檢測方法 14
2.3.1 生化測定菌種 15
2.3.2 PCR原理及檢測方法介紹 15
2.3.4 Analytical profile index(API)system 17
2.3.5 Vitek system 18
2.3.6 Chromogenic agar plate 19
2.3.7 免疫分析法(Immunoassay) 20
第三章 實驗方法與步驟 28
3.1 實驗目標 28
3.2 實驗架構 29
3.3 樣本收集 31
3.4 設計探針 33
3.5 修飾硫醇DNA固定在金奈米粒子表面 34
3.6 膜上固定Anti-Avidin Antibody 35
3.7 核酸純化 36
3.8 核酸定量 37
3.9 免疫層析法偵測沙門氏菌 38
3.10 Vitek2 39
3.11 藥品設備 40
3.12 實驗儀器 41
第四章 結果與討論 42
4.1 金奈米粒子免疫層析法偵測沙門氏菌雙股DNA 42
4.1.1 偵測沙門氏菌單一菌落culture sample 42
4.1.2 免疫層析法偵測沙門氏菌雙股DNA之偵測極限 44
4.2 金奈米粒子免疫層析法之臨床檢測 45
4.2.1 臨床檢測 45
4.2.2 臨床檢測之數據分析 47
第五章 結論 49
參考文獻 51
附錄 56
論文參考文獻:[1] Bendayan, M. (2001). "Worth its weight in gold." Science, 291(5507): 1363.
[2] Penn, S. G., L. He, et al. (2003). "Nanoparticles for bioanalysis." Current Opinion in Chemical Biology ,7(5): 609-615.
[3] 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.
[4] Clark, M. A. and E. L. Barrett (1987). "The phs gene and hydrogen sulfide production by Salmonella typhimurium." Journal of bacteriology, 169(6): 2391.
[5] Doyle, M. P. (1989). Foodborne bacterial pathogens, CRC.
[6] Cook et al., (2003). "Conventional Methods versus 16S Ribosomal DNA Sequencing for Identification of Nontuberculous Mycobacteria: Cost Analysis " J Clin Microbiol, 41 (3) , 1010-1015
[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] 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.
[9] D'Aoust, J. Y. (1991). "Pathogenicity of foodborne Salmonella." International Journal of Food Microbiology, 12(1): 17-40.
[10] Dinesh Prasad , Shankaracharya , Ambarish Sharan Vidyarthi. (2011) "Gold nanoparticles-based colorimetric assay for rapid detection of Salmonella species in food samples. " World J Microbiol Biotechnol, 27, 2227–2230
[11] Doyle, M. P., L. R. Beuchat, et al. (1997). Food microbiology: fundamentals and frontiers, ASM Press Washington, DC.
[12] E.Galikowska, et al. (2011). “Specific detection of Salmonella enteric and Escherichia coli strain by using ELISA with bacteriophages as recognition agents” Eur J clin Microbial Infect Dis 30:1067-1073
[13] Gaillot, O., P. Di Camillo, et al. (1999). "Comparison of CHROMagar Salmonella medium and Hektoen enteric agar for isolation of salmonella from stool samples." Journal of clinical microbiology 37(3): 762.
[14] 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 salmonella from food products." Applied and environmental microbiology 26(3): 288.
[15] Holmes, B., W. Willcox, et al. (1978). "Identification of Enterobacteriaceae by the API 20E system." Journal of Clinical Pathology 31(1): 22.
[16] Jain, K. K. (2005). "Nanotechnology in clinical laboratory diagnostics." Clinica Chimica Acta 358(1-2): 37-54.
[17] Joshi, R., Janagama, H., Dwivedi, H.P., Senthil Kumar, T.M., Jaykus, L.A., Schefers, J.,Sreevatsan, S.(2009) "Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars. " Mol. Cell. Probes.23, 20–28.
[18] 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).
[19] Katz, E. and I. Willner (2004). "Integrated nanoparticle: Vbiomolecule hybrid systems:synthesis, properties, and applications." Angewandte Chemie International Edition43(45): 6042-6108.
[20] Keum-Il Jang, et al. (2011). “Rapid and Simple Biochemical Detection for Salmonella spp. Using Modified LB Broth and the MUCAP Test” Food Sci. Biotechnol. 20(1): 201-207
[21] Kumar, S., K. Balakrishna, et al. (2005). "Rapid detection of Salmonella typhi in foods bycombination of immunomagnetic separation and polymerase chain reaction."World Journal of Microbiology and Biotechnology 21(5): 625-628.
[22] Le Minor, L. and M. Y. Popoff (1987). "Designation of Salmonella enterica sp. nov., nom. rev., as the Type and Only Species of the Genus Salmonella: Request for an Opinion." International Journal of Systematic and Evolutionary Microbiology, 37(4): 465.
[23] Malorny, B., E. Paccassoni, et al. (2004). "Diagnostic real-time PCR for detection of Salmonella in food." Applied and environmental microbiology 70(12): 7046.
[24] Mirkin, C. A., R. L. Letsinger, et al. (1996). "A DNA-based method for rationally assembling nanoparticles into macroscopic materials."
[25] Murray, P. R., W. L. Drew, et al. (1990). Medical microbiology, Wolfe Medical Publications Ltd.
[26] 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.
[27] Prescott, L., J. Harley, et al. (2002). "Microbiology, 5th." McGraw Hil1.
[28] Ruiz, J., M. L. Nunez, et al. (1996). "Comparison of five plating media for isolation of Salmonella species from human stools." Journal of clinical microbiology 34(3): 686.
[29] Ryan, K. J., C. G. Ray, et al. (2004). Sherris medical microbiology: an introduction to infectious diseases, McGraw-Hill Medical.
[30] Salehi, T. Z., M. Mahzounieh, et al. (2005). "Detection of InvA gene in isolated Salmonella from broilers by PCR method." Int. J. Poult. Sci 4(8): 557:V559.
[31] 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.
[32] Stewart, M. E., C. R. Anderton, et al. (2008). "Nanostructured plasmonic sensors." Chemical Reviews 108(2): 494-521.
[33] 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.
[34] 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.
[35] Tirado, C., & Schmidt, K. (2001). "WHO Surveillance Programme for control of foodborne infections and intoxications: Preliminary results and trends across greater Europe. "Journal of Infection, 43, 80–84.
[36] Wallet F., C. Loı¨ez, E. Renaux, N. Lemaitre, R. J. Courcol, (2005) "Performances of VITEK 2 Colorimetric Cards for Identification of Gram-Positive and Gram-Negative Bacteria" Journal of Clinical Microbiology, 43, 4402–4406.
[37] Wilson, R. (2008). "The use of gold nanoparticles in diagnostics and detection." Chem.Soc. Rev. 37(9): 2028-2045.
[38] 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.
[39] Zhouping Wang, et al. (2011). ” Sensitive detection of Salmonella with fluorescent bioconjugated nanoparticles probe” Food Chemistry 125: 779–784
[40] 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. Colony-printing gold-nanoparticle immunoassay in detection of Salmonella spp. isolated on media", 台北科技大學.
[41] 陳柏豪 (2011). "金奈米免疫層析法偵測沙門氏菌. Rapid detection of Salmonella using gold nanoparticle and immunochromatography", 台北科技大學.
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