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論文中文名稱:利用奈米金粒子結合拓印法來偵測培養皿上的沙門氏菌 [以論文名稱查詢館藏系統]
論文英文名稱:Colony-printing gold-nanoparticle immunoassay in detection of Salmonella spp. isolated on media [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
出版年度:97
中文姓名:曾瑋佑
英文姓名:Wei-Yu Tzeng
研究生學號:95688007
學位類別:碩士
語文別:中文
口試日期:2008-07-15
論文頁數:135
指導教授中文名:侯劭毅
指導教授英文名:Shao-Yi Hou
口試委員中文名:黃榮堂;黃聲東;王勝仕
中文關鍵詞:沙門氏菌奈米金粒子抗體拓印法
英文關鍵詞:Salmonellagold-nanoparticleantibodyColony-printing
論文中文摘要:本研究主要是發展一套非PCR類的、並且能夠快速偵測細菌的方法;這樣較快速的方法能夠及早發現病原菌的存在並且幫助臨床醫生做出適當的決策。所以在本實驗中,我們利用接有抗沙門氏菌抗體奈米金粒子以及拓印法兩者結合以應用於偵測醫院臨床上的樣本中,是否含有沙門氏菌存在。拓印法利用硝化纖維膜在長有沙門氏菌及其它的菌種的瓊脂平板上,在一定範圍內輕輕沾粘菌落,如此一來,膜上除了黏有菌外,各種菌落的相對位置也被記錄了下來,之後再將此膜浸泡於接有沙門氏菌抗體、紅色奈米金粒子溶液中,結果欲偵測的菌種就會在膜上顯示出顏色,其它菌種則不會呈現顏色,之後觀察膜上有顏色的部份,利用其相對位置,比照一下瓊脂平板上生長的菌落,即可知哪一個菌落是屬於沙門氏菌的菌。本研究從2007年9月到2008年4月從台北馬偕醫院收集病人檢體,總數為577個,將樣本培養於HE plate上並做檢測;測試結果與Vitek 2測試結果做比較,發現本研究檢測技術靈敏度可高達100%,專一性高達99%左右。
論文英文摘要:This study is to develop a non-PCR method for early detection of bacteria which can lead to help clinical physicians in making proper decisions. Different bacteria were spread on agar plates and then incubated to form colonies. A dry nitrocellulose paper was used to gently cover the agar surface by a one-touch press only by the paper’s own weight. The protocol was based on the transfer of surface cells of the colonies to the nitrocellulose membrane. Gold nanoparticles coated with the anti-Salmonella antibody were used to highlight the Salmonella on the membrane to facilitate the selectivity. The red dots on the membrane indicate the existence of Salmonella because gold nanoparticles are red instead of golden. From September 2007 to April 2008, 577 stool samples from patients at Mackay Memorial Hospital, Taipei were tested on Hektoen enteric agar plate. The black or crystalloid colonies were tested using this method. Compared with Vitek 2, the sensitivity of this new method is 100% and the specificity is about 99%.
論文目次:目 錄

中文摘要 i
英文摘要 ii
誌謝 iv
目錄 v
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 研究背景與原理介紹 5
2.1 病源菌的危害 5
2.2 現行Salmonella(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)原理介紹 11
2.3 Analytical profile index (API)system 11
2.4 Vitek system 15
2.5 Chromogenic agar plate 17
2.6 免疫分析法(Immunoassay) 21
第三章 實驗方法與步驟 24
3.1 實驗目標 24
3.2 實驗架構 24
3.3 樣本收集 26
3.4 Gold-nanoparticle immunoassay(奈米金免疫分析)介紹 26
3.5 Colony-printing(菌落拓印法)介紹 29
3.6 檢體樣本收集 32
3.7 Quality one軟體分析 34
3.8 實驗藥品與設備 35
3.8.1 實驗藥品 35
3.8.2 實驗設備 36
第四章 實驗結果 37
4.1 抗體奈米金粒子免疫分析實驗 37
4.2 Colony-printing(菌落拓印法)實驗 38
4.3 檢測臨床病人檢體有無沙門氏菌存在 41
4.4 使用Quality one軟體分析實驗結果 46
第五章 結論 53
5.1 抗體接於奈米金粒子實驗討論 53
5.2 Colony-printing(菌落拓印法)實驗討論 54
5.3 檢測所使用之培養基 55
5.4 菌種鑑定 56
參考文獻 58
附錄 63
附錄一 所有檢測樣本為Salmonella,其使用Quality one分析結果圖。
64
附錄二 所有檢測樣本為E.coli,其使用Quality one分析結果圖。 76
附錄三 所有檢測樣本為Citrobacter freundii,其使用Quality one分析結
果圖。 100
附錄四 所有檢測樣本為Proteus mirabilis,其使用Quality one分析結
果圖。 110
附錄五 所有檢測樣本為Morganella morganii,其使用Quality one分析結
果圖。 118
附錄六 所有檢測樣本為Citrobacter youngae,其使用Quality one分析結
果圖。 122
附錄七 所有檢測樣本為Peudomonas aeruginosa,其使用Quality one分析
結果圖。 125
附錄八 所有檢測樣本為Proteus vulgaris,其使用Quality one分析結果
圖。 127
附錄九 所有檢測樣本為Citrobacter amalonaticus,其使用Quality one分
析結果圖。 128
附錄十 所有檢測樣本為Citrobacter koseri,其使用Quality one分析結果
圖。 129
附錄十一 所有檢測樣本為Vibrio parahaemolyticus,其使用Quality one分
析結果圖。 130
附錄十二 所有檢測樣本為Plesiomonas shigelloides,其使用Quality one分
析結果圖。 131
附錄十三 所有檢測樣本為Shigella flexneri,其使用Quality one分析結果
圖。 132
附錄十四 所有檢測樣本為Citrobacter braakii,其使用Quality one分析結
果圖。 133
附錄十五 所有檢測樣本為Klebsiella pneumoniae,其使用Quality one分析
結果圖。 134



表目錄

表2.1 PCR偵測細菌的相關文獻整理 10
表2.2 API檢驗系統的相關文獻整理 14
表2.3 Vitek檢驗系統相關文獻整理 16
表2.4 Chromogenic agar plate偵測細菌的相關文獻整理 20
表2.5 奈米粒子利用來偵測物質相關文獻整理 23
表3.1 實驗藥品 35
表3.2 實驗設備 36
表4.1 未使用Quality one分析得到的統計結果 45
表4.2 所有菌種及其強度密度值平均 47
表4.3 所有菌種其附錄對照號碼表 48
表4.4 使用Quality one分析之後所得的統計結果 52






















圖目錄

圖2.1 Strip的結構圖 12
圖2.2 API-20E strip的一般檢驗流程 13
圖2.3 生長在HE plate上菌落顏色圖 18
圖2.4 沙門氏菌Salmonella生長在HE plate上菌落顏色圖 19
圖2.5 奈米金溶液圖 22
圖3.1 整體實驗流程圖 25
圖3.2 抗體接金簡圖 27
圖3.3 Gold-nanoparticle immunoassay作用原理示意圖 28
圖3.4 Colony-printing操作流程簡圖 30
圖3.5 Colony-printing(菌落拓印法)操作檢驗流程圖解 31
圖3.6 Stool culture實際情形 33
圖3.7 使用Quality one實際分析情形,及分析公式 34
圖4.1 抗體奈米金粒子免疫分析結果 37
圖4.2 對塗有三種菌的LB agar plate上某特定範圍做拓印及其檢測結果 39
圖4.3 對塗有三種菌的Hektoen enteric agar plate上某特定範圍做拓印及
其檢測結果 40
圖4.4 檢測臨床病人檢體結果情形(true negative) 42
圖4.5 檢測臨床病人檢體結果情形(true positive) 43
圖4.6 檢測臨床病人檢體結果情形(false positive) 44
圖4.7 所有分析樣本的強度密度情形。圖中可以發現檢測結果為Salmonella
的部份其強度密度值普遍高於其它樣本的強度 49
圖4.8 使用Quality one分析結果(negative but with red dot)。 50
圖4.9 使用Quality one分析結果(true positive) 51
論文參考文獻:1. Aguirre P. M, J. B. Cacho, L. Folgueira, M. Lopez, J. Garcia, and A. C. Velasco, 1990, “Rapid Fluorescence Method for Screening Salmonella spp. from Enteric Differential Agars”, Journal of Clinical Microbiology, 28, 148–149.
2. Chiu T. H., T. R. Chen, W. Z. Hwang, and H. Y. Tsen, 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, 259–265.
3. Cudjoe K. S., T. Hagtvedt, and R. Dainty, 1995, “Immunomagnetic separation of Salmonella from foods and their detection using immunomagnetic particle(IMP) -ELISA”, International Journal of Food Microbiology, 27, 11–25.
4. Cooke V. M, R. J. Miles, R. G. Price, and A. C. Richardson, 1999, “A Novel Chromogenic Ester Agar Medium for Detection of Salmonellae”, Applied and Environmental Microbiology, 65, 807–812.
5. Castro N. C., E. Gotuzzo, M. Rodriguez, and H. Guerra, 2000, “Clinical Application of a Dot Blot Test for Diagnosis of Enteric Fever Due to Salmonella enterica Serovar Typhi in Patients with Typhoid Fever from Colombia and Peru”, Clinical and Dlagnostic Laboratory Immunology, 7, 312–313.
6. Dusch H., and M. Altwegg, 1993, “Comparison of Rambach Agar, SM-ID Medium, and Hektoen Enteric Agar for Primary Isolation of Non-typhi Salmonellae from Stool Samples”, Journal of Clinical Microbiology, 31, 410–412.
7. Gaillot O., P. D. Camillo, P. Berche, R. Courcol, and C. Savage, 1999, “Comparison of CHROMagar Salmonella Medium and Hektoen Enteric Agar for Isolation of Salmonellae from Stool Samples ”, Journal of Clinical Microbiology, 37, 762–765.
8. Holmes B., W. R. Willcox, and S. P. Lapage, 1978, “Identification of Enterobacteriaceae by the API 20E system”,Journal of Clinical Pathology, 31, 22–30.
9. Joyanes P., M. D. C. Conejo, L. M. Martinez, and E. J. Perea, 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, 3247–3253.
10. Karami A, R. Ranjbar, Z. Ahmadi, and Z. Safiri, 2007, “Rapid Detection of Different Serovares of Salmonella entrica by Multiplex PCR”, Iranian J Publ Health, 36, 38–42.
11. Kumar S, K. Balakrishna, G.P. Singh, and H.V. Batra, 2005 “Rapid detection of Salmonella typhi in food by combination of immunomagnetic separation and polymerase chain reation”, World Journal of Microbiology & Biotechnology, 21, 625–628.
12. Kelly S., M. Cormican, L. Parke, G. C. Feeney, and J. Flynn, 1999, “Cost-Effective Methods for Isolation of Salmonella enterica in the Clinical Laboratory”, Journal of Clinical Microbiology, 37, 3369.
13. Kumar S., K. Balakrishna, G. P. Singh, and H. V. Batra, 2005, “Rapid detection of Salmonella typhi in foods by combination of immunomagnetic separation and polymerase chain reaction”, World Journal of Microbiology & Biotechnology, 21, 625-628.
14. Kulagina N. V., K. M. Shaffer, G. P. Anderson, F. S. Ligler, and C. R. Taitt, 2006, “Antimicrobial peptide-based array for Escherichia coli and Salmonella screening”, Analytica Chemica Acta, 575, 9–15.
15. Lowe P., H. Haswell, and K. Lewis, 2006, “Use of Various Common Isolation Media To Evaluate the New VITEK 2 Colorimetric GN Card for Identification of Burkholderia pseudomallei”, Journal of Clinical Microbiology, 44, 854–856.
16. Malorny B., E. Paccassoni, P. Fach, Cornelia Bunge, Annett Martin, and Reiner Helmuth, 2004, “Diagnostic Real-Time PCR for Detection of Salmonella in Food”, Applied and Environmental Microbiology, 70, 7046-7052.
17. Maddocks S., T. Olma, and S. Chen, 2002, “Comparison of CHROMagar Salmonella Medium and Xylose-Lysine-Desoxycholate and Salmonella-Shigella Agars for Isolation of Salmonella Strains from Stool Samples”, Journal of Clinical Microbiology, 40,2999–3003.
18. Mcnaughton B. H., R. R. Agayan, R. Clarke, R. G. Smith, and R. Kopelman, 2007, “Single bacterial cell detection with nonlinear rotational frequency shifts of driven magnetic microspheres”, Applied Physics Letters, 91, 224105-1— 224105-3.
19. Nye K J, D. Fallon, D. Frodsham, B. Gee, C. Graham, S. Howe, S. Messer, and T. Turner, 2002, “An evaluation of the performance of XLD, DCA, MLCB, and ABC agars as direct plating media for the isolation of Salmonella enterica from faeces”, J Clin Pathol, 55, 286–288.
20. Nie S., Y. Xing, G. J. Kim, and J. W. Simons, 2007, “Nanotechnology Applications in Cancer”, Review in Advance, 9, 14–50.
21. Pathmanathan S. G., N. C. Castro, M. M. S. Jime´nez, M. M. C. Ochoa, S. D. Puthucheary, and K. L. Thong, 2003, “Simple and rapid detection of Salmonella strains by direct PCR amplification of hilA gene”, Journal of Medical Microbiology, 52, 773–776.
22. Perez J. M., P. Cavalli, C. Roure, R. Renac, Y. Gille, and A. M. Freydiere, 2003, “Comparison of Four Chromogenic Media and Hektoen Agar for Detection and Presumptive Identification of Salmonella Strains in Human Stools ”, Journal of Clinical Microbiology, 41, 1130–1134.
23. Prescott L. M., J. P. Harley, and D. A. Klein,2005,“Microbiology”,Sixth edition,McGRAW.HILL,Boston.
24. Pascual M., M. Hugas, J. I. Badiola, J. M. Monfort, and M. Garriga, 1999, “Lactobacillus salivarius CTC2197 Prevents Salmonella enteritidis Colonization in Chickens”, Applied and Environmental Microbiology, 65, 4981–4986.
25. Ruiz J., M. L. N. Ez, J. N. Az, I. Lorente, J. N. P.Rez, and J. G.Mez, 1996, “Comparison of Five Plating Media for Isolation of Salmonella Species from Human Stools”, Journal of Clinical Microbiology, 34, 686–688.
26. Santos L. R. D., V. P. D. Nascimento, S. D. D. Oliveira., M. L. Flores, A. P. Pontes, R. Ribeiro, C. T. P. Salle, and R. F. F. Lopes, 2001, “Polymerase Chain Reaction(PCR)for Detection of Salmonella in Artificially Inculated Chicken Meat”, Revista Do Instituto De Medicina tropical De Sao Paulo, 43, 247–250.
27. Salman H. H., C. Gamazo, M. A. Campanero, and J. M. Irache, 2005, “Salmonella-like bioadhesive nanoparticles”, Journal of Controlled Release, 106, 1–13.
28. Steingroewer J., H. Knaus, T. Bley, and E. Boschke, 2005, “A Rapid Method for Pre-Enrichment and Detection of Salmonella Typhimurium by Immunomagnetic Separation and Subsequent Fluorescence Microscopical Techniques”, Engineering in Life Science, 5,267–272.
29. Salehi T. Z., M. Mahzounieh, and A. Saeedzadeh, 2005, “Detection of InvA Gene in Isolated Salmonella from Broilers by PCR Method”, International Journal of Poultry Science, 8 ,557-559.
30. Smith P. B., K. M. Tomfohrde, D. L. Rhoden, and A. Balows, 1972, “API System: a Multitube Micromethod for Identification of Enterobacteriaceae”, Applied Microbiology, 24, 449–452.
31. Thomas K. W. L., P. C. Tam, Z. K. Liu, and A. F. B. Cheng, 2001, “Evaluation of VITEK 2 Rapid Identification and Susceptibility Testing System against Gram-Negative Clinical Isolates”, Journal of Clinical Microbiology, 39, 2964–2966.
32. Valanne A., S. Huopalahti, T. Soukka, R. Vainionpaa, T. Lovgren, and H. Harma, 2005, “A sensitive adenovirus immunoassay as a model for using nanoparticle label technology in virus diagnostics”, Journal of Clinical Virology, 33, 217–223.
33. Wallet F., C. Loı¨ez, E. Renaux, N. Lemaitre, and 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.
34. Zhao X., L. R. Hilliard, S. J. Mechery, Y. Wang, R. P. Bagwe, S. Jin, and W. Tan, 2004, “A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles”, PNAS, 101, 15027–15032.
35. 陳錫圭,2006, “以奈米金粒子為標記物之半抗原分子檢測”,國立台北科技大學生物科技研究所碩士論文。
36. 蔡文成,2003,“臨床微生物診斷學”,第九版,九州圖書文物有限公司,台灣台北。
論文全文使用權限:同意授權於2008-10-01起公開