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研究生:曾鈺婷
研究生(外文):Yu-Ting Tseng
論文名稱:金奈米粒子免疫色層分析法偵測沙門氏菌之臨床試驗
論文名稱(外文):Detection of Salmonella in the clinical samples using gold nanoparticle and immunochromatography
指導教授:侯劭毅侯劭毅引用關係
口試委員:王勝仕黃光策黃志宏
口試日期:2012-07-27
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:61
中文關鍵詞:免疫層析法沙門氏菌金奈米粒子16S rDNA
外文關鍵詞:Salmonella16S rDNAImmunochromatographyGold nanoparticle
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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。

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.

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


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