乳品病原性微生物生物晶片之開發 陳信硯、李世傑
E-mail: [email protected]
摘 要
乳品是營養價值很高的食品，市面上有許多的乳品深受消費者的喜愛。然而，乳牛成長的區域到榨乳與製成商品的工廠，
皆可能有多種人畜共通且與環境汙染相關的病原性微生物存在，而這些乳品微生物所造成的經濟損失很大；新鮮度又是乳 品類講求的重點，從製程到上市的時間非常短，因此發展快速準確的鑑定方法亦是非常重要的。本研究的目的在於針對容 易造成乳品品質危害之微生物Brucella spp.、Acinetobacter spp.、Pseudomonas spp.、Aeromonas spp.、Enterobacter spp.
、Moraxella catarrhalis和Pasteurella multocida進行PCR引子組與DNA探針的設計，初步完成可同時檢測數種影響乳品品質 之病原性與環境微生物屬的生物晶片，並且沒有交叉雜合的反應出現。未來只要將欲偵測的特異性探針加入即可擴增鑑定 之菌屬。 另外，食品中毒案件發生的原因很多，但其形成和危害常與食因性病原微生物息息相關。其中常見的包含 有Salmonella 屬、Staphylococcus aureus、Listeria monocytogenes及Escherichia coli等菌株。食品中毒案件之中通常具有一種 以上的病原菌存在，普遍又因環境存在相關之病原菌間接汙染到食品本身。因此本研究測試了經由人工方式所污染的食品
，在PCR靈敏度試驗方面，未經預培養直接檢測下，訊號約可達到N×103~104 cfu/ ml，並利用multiplex PCR搭配本研究 所建立的檢測晶片雛型進行雜合實驗。 依照圖譜結果顯示，可成功區分出Listeria monocytogens、Staphylococcus aureus
、Streptococcus agalactiae、Enterobacter sakazakii、Escherichia coli O157:H7、Vibrio parahaemolyticus、Salmonella spp.以 及Pseudomonas fluorescens，即使在其他非目標菌株的存在之下，不論是對於單一或是混和菌株的試驗中，都可以得到不受 干擾的特異性訊號，此一生物晶片系統的建立，可用於食品上對於常見之病原菌的例行檢測，達到快速又準確的檢測結果
。
關鍵詞 : 乳品、病原性微生物、生物晶片
目錄
目錄 封面內頁 簽名頁 授權書... iii 中文摘要... iv 英文摘 要... vi 誌謝 ... ix 目錄 ... x 圖目 錄... xiv 表目錄... xvi 1. 文獻整理... 1 1.1 病原菌之簡介... 1 1.1.1 布魯氏桿菌屬... 1 1.1.2 假單胞菌
屬... 1 1.1.3 產氣單腸菌屬... 2 1.1.4 不動桿菌屬... 2 1.1.5 腸桿菌屬... 3 1.1.6 出血敗血性巴斯德氏菌... 4 1.1.7 黏膜炎莫拉 菌... 4 1.1.8 單核球增多性李斯特菌... 5 1.1.9 無乳鏈球菌... 6 1.1.10 大腸桿菌... 7 1.1.11 腸炎弧菌... 8 1.1.12 金黃色葡萄球 菌... 8 1.1.13 沙門氏菌屬... 9 1.2 應用於食品病原菌檢測之分子生物學方法...
9 1.2.1 聚合?鏈鎖反應及其應用... 10 1.2.2 多套式聚合?鏈鎖反應及其應用... 10 1.2.3 免疫檢測法... 11 1.2.4 DNA探針分析法... 11 1.2.5 免疫磁珠分離
法... 12 1.3 生物晶片... 13 1.3.1 生物晶片的分類... 13 1.3.2 生物晶片的應用... 15 2. 乳品病原性與環境微生物屬生物晶片之開發... 16 2.1 前
言... 16 2.2 材料與方法... 17 2.2.1 實驗材料... 17 2.2.1.1 菌種... 17 2.2.1.2 培養基... 17 2.2.1.3 藥
品... 18 2.2.1.4 緩衝液及試劑... 18 2.2.1.5 儀器... 19 2.2.2 實驗方法... 20 2.2.2.1 PCR引子組以及寡核?酸探針設計... 20 2.2.2.2 PCR 引子組及寡核?酸探針之合成... 20 2.2.2.3 聚合?鏈鎖反應... 20 2.2.2.3.1 DNA之製
備... 21 2.2.2.3.2 不同菌屬之PCR特異性試驗... 21 2.2.2.4 多套式聚合?鏈鎖反特異性試驗... 22 2.2.2.5 各特異性引子組於食品檢測之應用... 22 2.2.2.5.1 鮮乳樣品的檢測應用... 22 2.2.2.6 寡核?酸生 物晶片之試驗... 23 2.2.2.6.1 寡核?酸探針之佈放... 23 2.2.2.6.2 雜交試
驗... 24 2.2.2.6.3 呈色反應... 24 2.2.2.7 以寡核?酸生物晶片進行特異性與靈 敏度試驗 25 2.3 結果與討論... 25 2.3.1 聚合?鏈鎖反應... 25 2.3.2 多套式 聚合?鏈鎖反應... 26 2.3.3 食品檢測之應用... 26 2.3.4 生物晶片雜合圖譜之試
驗... 27 2.4 結論... 28 3. 重要食品中毒病原性與環境微生物生物晶片之開發... 30
3.1 前言... 30 3.2 材料與方法... 31 3.2.1 實驗材
料... 32 3.2.1.1 實驗菌株... 32 3.2.1.2 培養基... 32 3.2.1.3 藥品... 33 3.2.1.4 緩衝液及試劑... 34 3.2.1.5 儀
器... 35 3.2.2 實驗方法... 35 3.2.2.1 PCR引子組特異性試驗...
35 3.2.2.2 食品檢測之應用... 36 3.2.2.2.1 特異性引子組應用於食品樣品之靈敏度試驗 36 3.2.2.2.2 多套式聚合?鏈鎖反應應用於食品之檢測. 37 3.2.2.3 以寡核?酸生物晶片進行特異性試驗... 39 3.3 結果與討論... 40 3.3.1特異性基因片段之PCR檢測... 40 3.3.2 食品檢測之應 用... 40 3.3.2.1 PCR引子組靈敏度試驗... 40 3.3.2.2 Multiplex PCR特異性試 驗... 41 3.3.2.3 Multiplex PCR靈敏度試驗... 41 3.3.2.4 晶片探針之特異性試驗...
42 3.3.2.5 晶片探針之靈敏度試驗... 42 3.4 結論... 43 4. 結
論... 44 參考文獻... 83 附錄... 98 參考文獻
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