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研究生:朱霈慈
研究生(外文):Pei-Tzu Chu
論文名稱:建立磁珠-奈米微脂球檢測技術平臺並應用於快速偵測牛奶中仙人掌桿菌之污染
論文名稱(外文):Development and comparison of three rapid immunomagnetic-bead separation systems for detecting Bacillus cereus in milk, based on different surface-modified immunoliposomal nanovesicles
指導教授:溫曉薇
指導教授(外文):Hsiao-Wei Wen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:80
中文關鍵詞:仙人掌桿菌免疫奈米微脂體免疫磁珠
外文關鍵詞:Bacillus cereusimmunoliposomal nanovesiclesimmunomagnetic beads
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仙人掌桿菌是台灣地區引起食物中毒的主要病原菌之一,感染時造成的中毒症狀包括嘔吐,發燒和腹瀉。為了保障消費者食的安全,本研究利用不同表面修飾的免疫奈米微脂體(IMLNs),建立了三種針對仙人掌桿菌快速且靈敏的免疫磁珠(IMBs)偵測平台。分析樣品時,先藉由在磁珠表面修飾上仙人掌桿菌抗體的免疫磁珠從樣品中分離出仙人掌桿菌,接著加入包埋螢光染劑的免疫奈米微脂體增加偵測訊號。在第一個檢測系統中,抗體直接地修飾在奈米微脂體表面;最適化結果為粒徑 300 nm 免疫微脂體上修飾 0.25 mol%的抗體。在牛奶樣品中,最低偵測極限(LOD)為 5 CFU/mL 仙人掌桿菌。而在第二個檢測系統中,將抗體標定在微脂體表面PEG的尾端上,以增加抗體和奈米微脂體的間距並增強其擺動性;而系統的最適化條件為 200 nm 的 PEG-IMLN上修飾0.5 mol% 抗體。在牛奶樣品中,第二個檢測平臺的LOD為 5 CFU/mL 的仙人掌桿菌。而為了降低檢測平臺在牛奶的LOD,我們進一步的設計了第三個檢測系統,以一種新型態的 PEG-IMLN,其表面修飾了1 mol% 的PEG和0.5 mol% 生物素。我們利用生物素標定的免疫微脂體檢測系統可成功的在 6小時內檢測出牛奶中的仙人掌桿菌,其LOD達到 2 CFU/mL。 為評估此三種檢測平臺的專一性,藉由測試九株革蘭氏陽性和陰性菌,結果顯示本實驗所建立的檢測平臺具有良好的專一性。本研究藉由不同表面修飾的免疫奈米微脂球發展出一種快速檢測牛奶當中仙人掌桿菌之方法。
Bacillus cereus is a major food-born pathogen in Taiwan and its major syndromes include vomiting, fever and diarrhea. To minimize the possibility of exposing consumers to pathogenic B. cereus, this study develops three rapid and sensitive assays that utilize immunomagnetic beads (IMBs) and different immunoliposomal nanovesicles (IMLNs) systems. In these assays, anti-B. cereus antibody-conjugated IMBs were applied to capture B. cereus in samples; fluorescent dyes-loaded IMLNs were employed to increase the detection signal. Hence, a sandwich complex was formed as “IMBs-B. cereus-IMLNs”. In the first system, antibodies were directly conjugated to the liposomal surface. The optimal IMLNs had a diameter of 300 nm conjugated 0.25 mol% antibodies. The limit of detection (LOD) of this first system was 5 CFU/mL of B. cereus in milk samples. For increasing the distance between antibodies and liposomal nanovesicles, the second system antibody was tagged on the far end of polyethylene glycol (PEG) chains on the liposomal surface. The optimal PEG-IMLN had a diameter of 200 nm conjugated 0.5 mol% antibodies. The LOD of this second assay was closed to the LOD of the first assay as 5 CFU/mL of B. cereus in milk. In order to decrease the LOD in milk, we further designed the third system, a new type of PEG-IMLN, which surface was modified by 1 mol% PEG chains and 0.5 mol% biotins. Successfully, the Biotin-PEG-IMLN detection system could reach to the 2 CFU/mL of B. cereus about 6 hours in milk. To evaluate the specificity of these assays, nine Gram positive and negative bacteria were tested and no significant interference was found. Conclusively, this study elucidates the feasibility of using a novel IMB/IMLN assay to detect less than 5 CFU/mL of B. cereus within one working day.
CONTENT
CHINESE ABSTRACT……………………………..……………………………….…........ I
ENGLISH ABSTRACT…………………………………………………………………........II
FIGURE CONTENT…………………………………………………………………............V
CHAPTER ONE: LITERATURE REVIEW…………………….…………………................1
1-1. Food Safety and Foodborne Diseases in Taiwan….…………………......1
1-2. Characteristics of Bacillus cereus……………………………………….......2
1-2-1. Pathogenicity of Bacillus cereus…………………………………………......2
1-2-2. Prevalence in Food………………………………………………………..........3
1-2-3. Methods for Detecting Bacillus cereus…………………………………......3
1-2-3-1. Culture-Based Methods………………………………………………….4
1-2-3-2. Animal Assay Methods…………………………………………………...4
1-2-3-3. Bioassay Methods………………………………………………………....5
1-2-3-4. Immunoassay Detection Methods……………………………………..5
1-2-3-5. Nucleic Acid-Based Assay……………………………………………….6
1-3. Liposomal Nanovesicles…………………………………………………........9
1-3-1. Compositions and Classifications of Liposomal Nanovesicles…………9
1-3-2. Surface Modification of Liposomal Nanovesicles………………………....10
1-3-3. Applications of Immunoliposomal Nanovesicles………………………….12
1-4. Research Approaches…………………………………………………….........12
CHAPTER TWO: MATERIALS and METHODS…………………………………… 17
2-1. Materials…………………………………………………………………. 17
2-2. Methods…………………………………………………………………... 18
2-2-1. Preparation of Immunomagnetic Beads (IMBs)………………………….. 20
2-2-2. Preparation of ‬Immunoliposomal Nanovesicles (IMLNs) ………………... 20
2-2-3. Preparation of ‬PEGylated-Immunoliposomal Nanovesicles (PEG-IMLNs) 22
2-2-4. Preparation of NeutrAvidin-tagged Liposomal Nanovesicles…………...... 24
2-2-5. Strains and their Growth Conditions…………………………………….... 24
2-2-6. Assay Performance ……………………………………………………….. 25
CHAPTER THREE: RESULT and DISCUSSION…………………………………... 27
3-1. Maximizing the Binding Capacity of Protein A Magnetic Beads‬…….. 27
3-2. Optimizing the Components of Wash Buffer‬………………………….. 28
3-3. Optimizing IMB Reaction Volume……………………………………… 29
3-4. Optimization of IMLNs…………………………………………………. 30
3-5. Optimization of PEG-IMLNs…………………………............................ 33
3-6. Optimization of NeutrAvidin-tagged Liposomal Nanovesicles and Biotin-tagged PEG-IMLN (Biotin-PEG-IMLNs)………………………35
3-7. Specific Analysis…………………………………………………………. 37
3-8. Assay Performance……………………………………………………….38
3-9. Comparison of three different assay systems on their detection ability....40
CHAPTER FOUR : CONCLUSION………………………………………………….. 42
REFERENCES………………………………………………………………………......69
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