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研究生:許瀞方
研究生(外文):ching-Fang Hsu
論文名稱:以複合式即時聚合酶鏈反應法偵測O157:H7型出血性大腸桿菌
論文名稱(外文):Detection of enterohemorrhagic Escherichia coli O157:H7 by using multiplex real-time PCR technology
指導教授:潘子明潘子明引用關係
指導教授(外文):Tzn-Ming Pan
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:106
中文關鍵詞:複合式即時聚合酶食品中毒菌鏈反應法O157:H7 型出血性大腸桿菌
外文關鍵詞:Escherichia coli O157:H7multiplex real-time PCR
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即時聚合酶鏈反應法目前被廣泛地應用在病原菌之檢驗上,本研究以複合式即時聚合酶鏈反應檢驗在培養基、糞便與食品檢體中的 O157:H7 型出血性大腸桿菌。本研究利用 2 對引子對及 2 條不同的螢光探針進行O157:H7型大腸桿菌中之目標基因偵測,包含了合成 O157 型體抗原基因 (rfbO157) 中之192 bp 片段及類志賀氏毒素第 2 型基因 (stx2) 中之 170 bp 片段。分析 217 各類菌株結果顯示,本研究根據特有的 O157 型體抗原基因以及類志賀氏毒素基因序列所設計之即時聚合酶鏈反應檢測法,能準確地檢測與區分出 O157:H7 型出血性大腸桿菌及非 O157 :H7大腸桿菌菌株,在檢測過程中,缺乏這二種基因之菌株,皆呈現陰性反應。此套即時聚合酶鏈反應方法,對於牛奶檢體不經過增殖即直接檢測時,能定量分析之濃度線性範圍為 103~109 CFU/mL,針對糞便檢體和蘋果汁檢體的定量分析之濃度線性範圍為 104~109 CFU/g (或104~109 CFU/mL),針對碎牛肉檢體的定量分析之濃度線性範圍為 105~109 CFU/g。牛奶及蘋果汁檢體經過增殖後再檢測時,能偵測範圍可達100~104 CFU/mL。本研究中針對 O157:H7 型出血性大腸桿菌合成 O157 型體抗原基因和類志賀氏毒素第 2 型之基因,進行即時聚合酶鏈反應,不僅能快速偵測存在糞便或食品材料檢體中的 O157:H7 型出血性大腸桿
菌,同時也可進行定量分析。
Real-time polymerase chain reaction (PCR) assays have been developed for detection and quantification of pathogens in recent years. A multiplex real-time PCR assay was designed and evaluated on the ABI 7700 sequence detection system (TaqMan®) to detect enterohemorrhagic Escherichia coli O157:H7 in pure culture, feces and food samples. Two sets of primers and fluorescent probes were used for amplification and real-time detection of a 192-bp region of the rfbO157 gene encoding E. coli O157:H7-specific O-antigen, and 170-bp segment of stx2 gene encoding Shiga-like toxin 2. Analysis of 217 bacterial strains demonstrated that the multiplex real-time PCR assay successfully distinguished E. coli O157:H7 serotype from non- E. coli O157:H7 serotypes and provided accurate profiling of genes encoding O-antigen and Shiga-like toxin 2. Bacterial strains lacking these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for the two genes were linear over DNA concentrations corresponding from 103 to 109 CFU/mL of E. coli O157:H7 in pure culture and milk sample. The real-time PCR allowed construction of standard curves that facilitated quantification of E. coli O157:H7 in feces, apple juice, milk and ground beef sample. Detection sensitivity of the real time PCR assay ranged from 104 to 109 CFU/g (or 104 to 109 CFU/mL) of feces or apple juice without enrichment. Detection sensitivity of the real time PCR assay ranged from 105 to 109 CFU/g of ground beef without enrichment.After enrichment of milk and apple juice samples in modified Tryptic Soy Broth, the detection of levels were from 100 to 104 CFU/mL. The real-time PCR assay for rfbO157 and stx2 proved to be a rapid test for detection of E. coli O157:H7 in food matrices and could also be used for quantification of E. coli O157:H7 in
foods or fecal samples.
目錄
中文摘要..................................................I
英文摘要.................................................II
目錄....................................................III
圖次.....................................................VI
表次...................................................VIII
縮寫表...................................................IX

第一章 前言...............................................1

第二章 文獻回顧...........................................3
2.1. 大腸桿菌..........................................3
2.1.1. 致病性大腸桿菌...............................6
2.1.2. 腸毒性大腸桿菌 ..............................6
2.1.3. 腸出血性大腸桿菌.............................7
2.1.4. 腸侵襲性大腸桿菌.............................8
2.1.5. 附著性大腸桿菌...............................8
2.1.6. 擴黏型大腸桿菌...............................8
2.2. Escherichia coli O157: H7............................12
2.2.1. 疫情........................................12
2.2.2. 基礎研究 (生化特性)、基因序列.................14
2.2.3. 致病因子....................................16
2.2.4. 引起症狀及診斷..............................21
2.2.5. 致病能力及傳染途徑..........................22
2.2.6. 相關法規....................................23
2.3. E. coli O157:H7之檢驗方式..........................24
2.4. E. coli O157:H7檢驗流程............................28
2.5. 即時聚合酶鏈反應.................................34

第三章 材料方法..........................................38
3.1. 材料.............................................38
3.1.1. 菌株........................................38
3.1.2. 試劑........................................38
3.2. 實驗儀器與設備...................................41
3.3. 方法.............................................43
3.3.1. DNA 之製備與純化...........................43
3.3.2. 引子和探針之設計............................45
3.3.3. 構築質體 (定量分析之標準參考物質) ...........45
3.3.4. 單一即時聚合酶鏈反應定性分析................47
3.3.5. 定量標準菌株質體之抽取......................48
3.3.6. 複合式即時聚合酶鏈反應定性及定量分析........49
3.3.7. 洋菜膠體電泳分析............................51
3.3.8. PCR 產物之定序及序列比對....................52
3.3.9. E. coli strains Tw02、Tw03 及 Tw04 之 stx2 基因定序..........................................52

第四章 結果與討論
4.1. E. coli O157:H7 Tw02、Tw03 及 Tw04 菌株之 stx2 基因定序...............................................59
4.2.引子對專一性測試結果..............................59
4.2.1. 單對式即時聚合酶鏈反應檢測系統之建立........65
4.2.2. 複合式即時聚合酶鏈反應檢測系統之建立........67
4.3. E. coli O157:H7 的定量分析系統的建立................71
4.4. 模擬食品檢體中E. coli O157:H7 之定性及定量結果.....73
4.4.1. 牛奶檢體....................................74
4.4.2. 模擬蘋果汁檢體..............................77
4.4.3. 模擬碎牛肉檢體..............................80
4.5. 模擬人類之糞便檢體中E. coli O157:H7 之定性及定量結果...............................................83
4.6. 模擬樣品檢驗結果.................................87
4.7. 含低濃度菌量檢體經增殖後之偵測結果...............89

第五章 結論..............................................91

第六章 參考文獻..........................................93
























圖次

圖 2-1 腸道病原性大腸桿菌其致病方式.............................................10
圖 2-2 培養在山梨醇-馬康克瓊脂平板培養基上之 E. coli O157:H7 菌落.............................................................................................15
圖 2-3 出血性大腸桿菌attaching-and-effecting作用圖.......................18
圖 2-4 EHEC 菌株之基因分佈圖.........................................................20
圖 2-5 美國疑似 E. coli O157 感染患者之糞便檢驗流程................30
圖 2-6 日本疑似感染 E. coli O157患者之糞便檢驗流程..................31
圖 2-7 日本疑似受 E. coli O157 污染之食品檢驗流程....................32
圖 2-8 台灣地區疑似感染 O157:H7 型大腸桿菌患者糞便之檢驗流程................................................................................................33
圖 2-9 鐵克曼探針(TaqMan Probe)之構造及作用方式..................37
圖 3-1 類志賀氏毒素基因第二型序列定序之示意圖.........................53
圖 4-1 E. coli O157:H7 Tw01、Tw02、Tw03 及 Tw04菌株所含之 stx2 基因序列。序列 1–4 分別為 E. coli Tw01 至 Tw04 菌株之序列..................................................................................................61
圖 4-2 以 E. coli O28 (stx2+、O157-)、E. coli O157:H7 ATCC43895 (stx2+、O157+)、E. coli O157:H7 ATCC43888 (stx2-、O157+) 及E. coli O78:H11 (stx2-、O157-) 進行stx2F 和 stx2R引子對專一性之初步測試結果................................................................63
圖 4-3 以 E. coli O28 (stx2+、O157-)、E. coli O157:H7 ATCC43895 (stx2+、O157+)、E. coli O157:H7 ATCC43888 (stx2-、O157+) 及O78:H11 (stx2-、O157-) 進行 rfbE.coliO157引子對專一性之初步測試結果....................................................................................69
圖 4-4 (a)由不同拷貝數之標準定量參考物質得到之 stx2基因表現量放大曲線;(b)由不同拷貝數之標準定量參考物質得到之回歸曲線............................................................................................72
圖 4-5 以熱破菌法萃取牛奶檢體中之 DNA 模板後,並以引子對
Ns5/Ns7 放大stx2 基因片段...................................................75
圖 4-6 (a)牛奶檢體之螢光放大曲線圖;(b)牛奶檢體之定量標準曲線圖................................................................................................76
圖 4-7 以改良之 Qiaamp DNA stool mini kit 萃取蘋果汁檢體中之 DNA 模板後,以引子對 Ns5/Ns7 放大 stx2 基因片段.......78
圖 4-8 (a)蘋果汁檢體之螢光放大曲線圖;(b)蘋果汁檢體之定量標準曲線圖..........................................................................................79
圖 4-9 以改良之Qiaamp DNA stool mini kit 萃取碎牛肉檢體中之 DNA 模板後,以引子對 Ns5/Ns7 放大 stx2基因片段.........81
圖 4-10 (a)人體碎牛肉檢體之螢光放大曲線圖;(b)人體碎牛肉檢體之定量標準曲線圖........................................................................82
圖 4-11 以 Qiaamp DNA stool mini kit 萃取人體糞便檢體中之 DNA 模板後,以引子對 Ns5/Ns7 放大 stx2 基因片段...............84
圖 4-12 (a)人體糞便檢體之螢光放大曲線圖;(b)人體糞便檢體之定量標準曲線圖................................................................................85
















表次

表 2-1 大腸桿菌之一般生化特性..........................................................4
表 2-2 為各種病原性大腸桿菌致病性以及症狀之比較.....................11
表 3-1 本研究使用之 O157 :H7 型大腸桿菌菌株編號及來源.........54
表 3-2 本研究使用之非 O157 :H7 型大腸桿菌之菌株編號.............55
表 3-3 本研究使用之非大腸桿菌之菌株編號.....................................57
表 3-4 本研究所使用之引子序列.........................................................58
表 4-1 E. coli Tw01~Tw04 之 stx2 基因序列相似度比對結果..........62
表 4-2 本研究所設計之引子 stx2F及 stx2R專一性之測試.............64
表 4-3 以複合式即時聚合酶鏈反應檢測本研究中所有菌株之結果.70
表 4-4 以複合式聚合酶鏈反應測試牛奶、蘋果汁以及人體糞便中
E. coli O157:H7 Tw04之靈敏度...............................................86
表 4-5 偵測盲樣檢體中之 E. coli O157:H7 Tw04...............................88
表 4-6 以複合式聚合酶鏈反應測試經增殖後的牛奶、蘋果汁以及人體糞便中E. coli O157:H7 Tw04之靈敏度...............................90
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