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研究生:洪秋蓮
研究生(外文):Cho-Lien Hung
論文名稱:以Oligonucleotidearray檢測沙門氏菌屬、大腸桿菌、葡萄球菌屬、仙人掌桿菌群及弧菌屬之可行性探討及應用
論文名稱(外文):Development and application of Oligonucleotide array for the specific detection of Salmonella spp., Escherichia coli, Staphylococcus spp., Bacillus cereus group and Vibrio spp.
指導教授:曾浩洋曾浩洋引用關係
指導教授(外文):Hau-Yang Tsen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:160
中文關鍵詞:寡核甘酸陣列16S rRNA基因病原菌
外文關鍵詞:oligonucleotide array16S rRNA genepathogen
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中文摘要
腸炎弧菌、金黃色葡萄球菌、仙人掌桿菌群、沙門氏菌、病原性大腸桿菌為造成台灣地區食品中毒之常見病原菌。傳統檢測法先利用選擇性與鑑別性培養基挑出可疑菌落,再進行生化試驗以及血清型鑑定等步驟,費力且費時,故發展一套快速檢測方法有其重要性。生物晶片即為目前先進的技術之一,由於16S rRNA基因已成為細菌鑑定及分類學之常見方法,因此本實驗擬發展一套以16S rRNA基因做為不同細菌檢測用之探針為設計基礎之生物晶片,以同時檢測多種不同病原菌。
本論文實驗結果,利用10條寡核苷酸探針,進行oligonucleotide array實驗,可以建立五種雜合型式 (hybridization patterns),用以區分沙門氏菌屬、大腸桿菌、仙人掌桿菌群、葡萄球菌屬及弧菌屬,於混合菌株之檢測也呈現預期之雜合型式。並發現有三組寡核苷酸探針 (Sal為Salmonella 特異性探針、Ecoli及16S-001為特異性探針),可以之另外作為PCR檢測之用途。於純菌之檢測,經增殖8小時後,其檢測靈敏度可達100 CFU/ per assay;於食品中目標細菌之應用檢測,於8小時預培養後,靈敏度尚不理想,且雜合型式不易判斷污染之細菌種類之現象。
本論文所發展之oligonucleotide array用於純菌之檢測,對於菌株之鑑定,具有良好之檢測結果,唯於食品之應用,仍待相關技術改善。
Abstract
Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus group, Salmonella spp., pathogenic Escherichia coli are common pathogens which may cause food poisoning cases. Conventional methods for the detection of these bacteria spp. need the use of selection and differentiation medium followed by biochemical and serological identification steps. Such process is time consuming and laborious. Thus, development of rapid methods for the detection of these bacteria species is important. Biochip may be one of the choices for such purpose. Owing to the fact that 16S rRNA gene sequences have been used for bacteria identification and classification, in this study we thus tried to develop a 16S rRNA gene based biochip for the simultaneous detection of several different species of food pathogens. We found that 10 oligonucleotides arrayed on a biochip could be used for the differentiation of 5 genus of pathogenic bacteria. These bacteria genus are Salmonella spp., E coli, Bacillus cereus group strains, Staphylococcus spp. and Vibrio spp. etc. Five distinct chip hybridization patterns could be obtained for these 5 genus of bacteria spp. In additions, the hybridization pattern allowed us to find three specific oligonucleotide probes which in combination with a universal primer, could be used for the PCR primers specific for the detection of Salmonella spp. and E. coli. For pure culture, after 8 hrs preculture step, the detection sensitivity for theses bacteria was 100 CFU/ assay. For target cells in food samples, however, detection was not sensitive enough even after 8 hrs preculture step. In conclusion, the oligonucleotide array could be used for the identification of pure cultured bacteria but in use of it for food inspection, the process need some improvements.
目 次
頁次
中文摘要……………………………………………………………………….1
英文摘要……………………………………………………………………….2
第一章 文獻整理
第一部分 沙門氏菌…………………………………………………………...4
第二部分 大腸桿菌…………………………………………………………...7
第三部分 仙人掌桿菌………………………………………………………...9
第四部分 金黃色葡萄球菌………………………………………………….15
第五部分 腸炎弧菌………………………………………………………….18
第六部分 分子生物學方法應用於食品病原菌之檢測…………………….20
(一) DNA探針分析法 (DNA probe method)..……………………...….20
(二) 免疫分析 (Immunoassay)……………………………………….….21
(三) 免疫磁珠分離法 (Immunomagnetic separation; IMS)…………….22
(四) 特異噬菌體 (Specificity-phage)…………………………………... 23
(五) 聚合酶鏈鎖反應 (Polymerase chain reaction) 及其應用…………23
(六) 多套氏聚合酶鏈鎖反應 (Multiplex-Polymerase chain reaction) 及其應用………………………………………………………………………...25
(七) 隨機擴增多形性DNA (Randomly Amplified Polymorphic DNA, RAPD)……………………………………………………………………...... 26
第七部分 生物晶片………………………………………………………….27
表 (1-1∼1-8) ………………………………………………………………...38
圖 (1-1∼1-4)………………………………………………………………....47
第二章 以oligonucleotide array檢測沙門氏菌屬、大腸桿菌、葡萄球菌屬、仙人掌桿菌群及弧菌屬及其應用
壹、 前言……………………………………………………………………...50
貳、 材料與方法……………………………………………………………...52
一. 實驗材料
(一) 菌種………………………………………………………………….52
(二) 藥品………………………………………………………………….52
(三) 培養基……………………………………………………………….53
(四) 緩衝液及試劑……………………………………………………….54
(五) 儀器………………………………………………………………….56
(六) 檢測樣品…………………………………………………………….57
二. 實驗方法
(一) PCR引子組以及寡核苷酸探針設計………………………………57
(二) PCR引子組及寡核苷酸引子之合成………………………………57
(三) 聚合酶鏈鎖反應 (PCR)……………………………………………57
1. DNA之製備…………………………………………………………..57
2. 以PCR增幅16S rRNA target之試驗………………………………..58
3. 寡核苷酸探針之PCR特異性試驗…………………………………..58
4. 以PCR增幅16S rRNA target之靈敏度試驗………………………..59
(四) oligonucleotide array之試驗………………………………………..59
1. 寡核苷酸探針之佈放……………………….………………………..59
2. 預雜合試驗 (prehybridization)及雜合試驗 (hybridization) ….…..59
3. 呈色試驗……………………….……………………………………..60
4. oligonucleotide array特異性之試驗…………..……………………..60
5. oligonucleotide array靈敏度之試驗……………….………………...60
6. 混合菌株之試驗………………….…………………………………..60
(五) 聚合酶鏈鎖反應 (PCR) 及oligonucleotide array於食品檢測之應用………………….……………………………………………………..61
1. 乳品之檢測應用………………………………….…………………..61
(1) 牛乳中生菌數之計算…………………………….…………………..61
(2) 牛乳中E. coli及Salmonella spp.之總菌數…….…………..………..61
(3) 直接檢測………………………………………….…………………..61
(4) 增菌培養………………………………………….…………………..61
2. 肉品之檢測應用………………………………….…………………..62
(1) 肉品中生菌數之計算…………………………….…………………..62
(2) 肉品中E. coli及Salmonella spp.之總菌數…….…………..………..62
(3) 直接檢測………………………………………….…………………..62
(4) 增菌培養………………………………………….…………………..62
參、 結果與討論…………………………………………....………………63
一. PCR引子組及寡核苷酸探針之設計…………………....………………63
二. 聚合酶鏈鎖反應 (PCR) ………………………………....…...…………63
1. DNA之製備……………………………………...…....…...…………64
2. 以PCR增幅16S rRNA target之試驗…………...…....…...…………64
3. 寡核苷酸探針之PCR特異性試驗…………...…....….…..…………65
4. 以PCR增幅16S rRNA target之靈敏度試驗…....….….....…………70
三. Oligonucleotide array pattern之試驗…....….…………….......…………71
1. Oligonucleotide array特異性之試驗結果…………….......…………71
2. 混合菌株之試驗結果…....….…………….....…………….…………74
3. Oligonucleotide array靈敏度之試驗結果…………….......…………74
四. Oligonucleotide array 應用於食品中目標菌之檢測…….......…………76
1. 乳品樣品之檢測…………………………………......…...………..…76
(1) 乳品樣品中生菌數、Salmonella 與E. coli之測定…...………..……77
(2) 應用於乳品中目標菌之直接檢測…...……………………..…..……77
(3) 增菌培養…………………………………......…...………………..…77
2. 肉品樣品之檢測…………………………………......…...………..…78
(1) 肉品樣品中生菌數、Salmonella 與E. coli之測定…...………..……78
(2) 應用於肉品中目標菌之直接檢測…...……………………..…..……78
(3) 增菌培養…………………………………......…...………………..…78
肆、 結論…………………………………………………………………... 80
表 (表2-1∼2-12) …………………………………………………………..81
圖 (圖2-1∼2-31) …………………………………………………………102
參考文獻 ……………………………………………………………………139
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