Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/56751
標題: 建立食品中仙人掌桿菌的三種奈米免疫微脂球快速檢測方式-奈米微脂球免疫側流層析試驗法(III)
Development of Three Different Rapid Assays for the Detection of Bacillus Cereus in Foods-Immunoliposomal Nanovesicle-Lateral Flow Assay (III)
作者: 溫曉薇
周鳳英
關鍵字: 基礎研究;Bacillus;食品科技;仙人掌桿菌;奈米微脂球;側流層析法;Cereus;Liposomal Nanovesicles;Lateral Flow Assay
摘要: 
根據衛生署統計,從民國80 年到95 年,台灣地區由仙人掌桿菌所引起的中毒案件占所有食品細菌中毒案件的11.8%,僅次於腸炎弧菌與沙門氏菌。由於仙人掌桿菌是廣泛存在於自然界當中,所以在食品工業製造的各個環節如:產地、加工廠、運輸、零售等,皆有可能受到此株菌體的污染,進而可能造成食品中毒的情況發生。所以本研究預計在三年中針對仙人掌桿菌研發出之三種不同的快速奈米微脂球免疫檢測方法,包含「奈米微脂球免疫吸附法」、「奈米微脂球免疫磁珠法」以及「奈米微脂球免疫側流層析法」,以適用於不同狀況下之食品中仙人掌桿菌的檢測。第一年計畫(2006.8~2007.7)的研究為建立「奈米微脂球免疫吸附法」,利用讀盤螢光儀將檢驗過程自動化,並可同時以96孔盤檢驗大量樣品,目前已達到在8 小時內可以104~103 CFU/ mL 的菌量,此研究仍朝向提高檢測靈敏度與降低作業時間等項目研發中。執行中之第二年計畫(2007.8~2008.7)的研究為建立「奈米微脂球免疫磁珠法」,利用磁珠粒徑小而可以充分與待測物混合的特性,以提高檢測的靈敏度,目前的結果顯示利用0.22-μm Durapore 膜做過濾濃縮後,在42℃下以TBS 培養基培養3 小時後加入奈米免疫微脂球進行偵測,其靈敏度可以到達102 CFU/ mL 的菌濃度,現階段正針對免疫磁珠法中的各步驟做最適化的調整,並將之應用於液態食品(牛奶或蘋果汁)的檢測。第三年計畫(2008.8~2009.7)為利用對仙人掌桿菌具有專一性辨識能力的多株抗體與具有呈色能力的奈米微脂球或膠態金屬顆粒作為檢驗試劑。當檢驗試劑與菌體結合會形成複合物,再將之與試紙上原先已固定上的抗仙人掌桿菌抗體結合,上以三明治模式進行反應,可產生肉眼可見的呈色結果。上述檢測方式為側流層析法。此技術的研發將著重在兩個部分:(1)最適當的材料選擇,包括試檢體墊、結合墊、薄膜、吸收墊及支撐板的種類。(2)檢驗試劑的各種最適化評估,包含有奈米微脂球及膠態金屬顆粒的尺寸大小、表面的抗體比例與適用的濃度與量。最後將所有最適化的結果製成側流層析試紙組,並將之實際進行在食品樣本(乳類製品、即食食品)或工廠及廚房食品處理機械表面的微生物檢驗等。今日在食品病原菌的檢測上,發展便宜、方便的檢驗技術而得到快速、準確的檢驗結果是主要的趨勢。與過去傳統的檢測方式比較,傳統的檢測方式是利用菌體的生化性質為原理進行檢測,非常耗時與費力。因「奈米微脂球免側流層析法」操作簡單且不需任何複雜儀器,故大幅縮短檢驗的時間並可應用於現場測試,希望藉此能減少食品污染與中毒的發生。

Between 1981 and 2005, 207 cases of Bacillus. cereus outbreaks were reported inTaiwan. This number represents 11.8% of total foodborne outbreaks, and ranks as the thirdplace, following Vibrio parahaemolyticus and Salmonella spp. B. cereus has been detected inmany parts of food manufactures, such as harvesting, processing, transportation, storage, andretails. These presences may finally cause contamination or food intoxication. The goal of thisproposal is to develop three rapid assays for detection of B. cereus in various samples,including immunoliposomal nanovesicle sandwich fluorescence (ILN/SF) assay,immunomagnetic bead immunoliposomal nanovesicle (IMB/ILN) fluorescent assay, andimmunoliposomal nanovesicle-lateral flow assay (ILN-LFA). In the 1st year (2006.8~2007.7),we focused on developing the ILN/SF assay by using microtiter plate reader. The detectioncan be automated and a large amount of samples can be analyzed simultaneously. Until now,the limit of detection (LOD) is 104~103 CFU/mL with the assay time as 8 hr. We are workingon improving the assay sensitivity and reducing the assay time. In the 2nd year(2007.8~2008.7), the objective is to develop an IMB/ILN assay for the detection of B. cereusin aqueous samples. To date, the 0.22-μm Durapore membrane is the best candidate forconcentrate bacteria, and the optimal enrichment condition is incubating in TSB at 42℃ for 3hours, with the LOD value as 103 CFU/mL. We will optimize each step of this assay, and thenapply the developed assay to detect B. cereus in aqueous samples, such as milk or apple juice.In the 3rd year project(2008.8~2009.7), anti-B. cereus antibody will be used toconjugate on the surface of liposomal nanovesicles or colloidal gold particles as the detectionreagents. Both liposomal nanovesicles and colloidal gold particles can provide the resultsvisually and instantly. The complex of “B. cereus-detection reagent” will be captured byanother anti-B. cereus antibody, which will be coated in the test line on the membrane.Subsequently, a red color band will appear to prove the presence of B. cereus in the testsample. This assay is named as lateral flow assay (LFA), which is a simplified version ofELISA. In this study, we will focus on two parts: (1) selecting the proper materials, includingmembrane, conjugate pad, sample pad, wicking pad, and backing; (2) optimizing theparameters of reagents, including the size and antibody mol% of liposomal nanovesicles andcolloidal gold particles, and the compositions of blocking buffer and coating buffer. Aftermanufacturing with the optimal conditions, this ILN-LFA will be applied to detect B. cereusin food samples or the surface of instruments. To date, it is important to develop rapid, simple,and low-cost screening assays for detecting foodborne pathogens. Conventional methods foridentifying B. cereus are time consuming and laborious. Since ILN-LFA is a quick and simplemethod without the need of delicate devices, it can do in-site test. Therefore, the frequentutilization of this assay may reduce the cases of B. cereus outbreak and financial loss.
URI: http://hdl.handle.net/11455/56751
其他識別: NSC97-2313-B005-018
Appears in Collections:食品暨應用生物科技學系

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