Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52209
標題: 台灣鮮切水果和蔬菜的細菌污染評估研究與奈米微脂球和二氧化矽奈米粒兩種螢光探針於仙人掌桿菌嘔吐毒素基因檢測平台之比較
An assessment study on bacterial contamination of fresh cut fruits and vegetables in Taiwan and comparison of liposomal nanovesicles and silica nanoparticles as fluorescent reportersfor the detection of the emetic toxin gene of Bacillus cereus
作者: 湯昭瑜
Tang, Jhao-Yu
關鍵字: 生鮮截切水果和蔬菜
fresh-cut fruits and vegetables
病原菌
總生菌數
大腸菌群
仙人掌桿菌毒素
奈米微脂球
二氧化矽奈米粒
核酸雜合反應檢測
pathogens
coliform
Bacillus cereus toxin
liposome nanovesicles
silica nanoparticles
nucleic acid hybridization detection
出版社: 食品暨應用生物科技學系所
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摘要: 第一部分: 微加工產品近幾年在台灣越來越受消費者的喜愛,因此即食生鮮截切水果和蔬菜產品可以輕易地購於台灣的零售商、便利店、大賣場、超市和餐館等場所。鮮切產品的特點是營養、方便、美味和新鮮。然而,在輕度加工生產過程中,可能增加微生物污染的風險。因此,調查評估即食食品的衛生品質是必要的。本研究針對20家公司的165件水果或蔬菜樣品進行了總生菌數,大腸菌群,李斯特菌,金黃色葡萄球菌,腸炎弧菌,沙門氏菌,產氣莢膜桿菌,大腸桿菌和仙人掌桿菌的汙染情形調查。在這項研究中,大腸菌群污染在即食產品最嚴重,不合格率為40%。共有7件樣品,分別是木瓜(D公司)、芭樂(H公司),牛番茄(L公司),萵苣(M公司),高麗菜(S公司)受到的沙門氏桿菌污染。而T公司的油菜產品則受到李斯特菌的污染,故台灣的此類相關企業需仍要提升產品加工過程中的衛生管控。 第二部分: 利用奈米微脂球和二氧化矽奈米粒兩種螢光探針建立仙人掌桿菌嘔吐毒素基因檢測平台並比較何者具有最好的偵測訊號。檢測方法是應用三明治型核酸雜合反應去偵測單股DNA序列。仙人掌桿菌的ces基因序列做為本研究的目標片段。將單股核苷酸探針利用生物素與抗生物素蛋白之間的親和性接合在磁珠表面。修飾上探針的奈米微脂球和二氧化矽奈米粒做為標記,利用DNA雜交反應標記在ces 序列上。藉由單股DNA與單股DNA的雜和反應,可以將目標序列分離出來,並偵測其標記上的螢光分子,推算出目標分子含量。分別找出兩種不同螢光探針在仙人掌桿菌嘔吐毒素基因檢測平台的最適化條件。其結果顯示在最佳檢測條件下,利用奈米微脂球螢光探針檢測仙人掌桿菌嘔吐毒素,擁有較高的螢光訊號、有較大的DNA檢測濃度範圍(3.3-100 nM),而計算出的檢測極限值為0.9 nM DNA。故本研究開發出一個高靈敏度的螢光奈米探針方法以檢測仙人掌桿菌ces基因。
Part I: Recently, the minimally-processed food products have been gradually popular in Taiwan. Fresh cut products can be purchased easily from retailers, convenience stores, hypermarkets, supermarkets, or instant restaurants. The characteristics of fresh-cut products are natural nutrition, handiness, savoriness and freshness. However, during processing, the minimally-processed fruits and vegetables are easily contaminated by bacteria. Therefore, investigating the amount of microbes especially pathogens in RTE food products becomes necessary. The study sampled a total of 165 minimally-processed fruits or vegetables from 20 companies to investigate their total plate count (TBC), coliform, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella spp., Clostridium perfringens, Escherichia coli, and Bacillus cereus. In this study, the most contaminated issue in RTE products were coliform since 40% of samples are unqualified. Moreover, there were 7 samples including papaya (Company D), guava (Company H), beef tomato (Company L), lettuce (Company M), and cabbage (Company S) which were contaminated by Salmonella spp. In addition, there was one sample, rape (Company T) which was contaminated by L. monocytogenes. Conclusively, the survey of this study shows that the companies need to improve their sanitary quality or the process of RTE and RTC products. Part II The liposomal nanovesicles and silica nanoparticles were used as fluorescent reporters for the detection of Bacillus cereus ces gene in a sandwich-type hybridization assay and their assay performance was compared in order to develop a sensitive assay with a lower detection limit. Oligonucleotide capturer pobes was conjugated to the surface of magnetic beads through an avidin–biotin linkage. The reporter DNA-tagged liposomes encapsulating SRB or silica nanoparticles were used as the label reagent to hybridize to DNA-tagged magnetic beads-ces sequence complex. Through a sandwich-type DNA hybridization procedure, the target DNA was captured and quantified by the fluorescent signals from liposomal nanovesicles or silica nanoparticles. The results of this study show that liposomal nanovesicle was a better detection reagent than silica nanoparticles and the assay with liposomal nanovesicles could have a dynamic range as 3.3-100 nM of target DNA with a detection limit of around 0.9 nM. Hence, this study has developed a probe tagged fluorescent nanovesicle method to detect B. cereus ces gene.
URI: http://hdl.handle.net/11455/52209
其他識別: U0005-2408201216261100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2408201216261100
Appears in Collections:食品暨應用生物科技學系

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