志賀氏菌與大腸桿菌malate dehydrogenase基因序列定序及檢測沙門氏菌之腸毒素基因與細胞毒性，並應用免疫磁珠分離法於多套式PCR同時檢測食品中之大腸桿菌O157:H7及沙門氏菌 ; Sequencing of the mdh genes of Shigella spp., E. coli, detection of enterotoxin gene and cytotoxicities for Salmonella spp. and the Simultaneous detection of E. coli O157:H7 as well as Salmonellae in foods using an Immunomagnetic separation-multiplex PCR

大腸桿菌是食品或環境遭受動物糞便污染之指標菌。病源性大腸桿菌會感染人類，造成疾病或食物中毒，因此就食品衛生安全而言，快速檢測方法受到重視；聚合酶鏈鎖反應法 (polymerase chain reaction; PCR)為其中方法之一。PCR或DNA probe應用於大腸桿菌的檢測上，由於志賀氏菌和大腸桿菌DNA同源性高，許多學者無法排除志賀氏菌之干擾，本研究室針對大腸桿菌之mdh基因設計PCR引子組，亦無法排除志賀氏菌之干擾。為了瞭解E. coli與Shigella spp.在mdh基因上的相似性，本實驗乃對志賀氏菌之mdh基因進行定序，發現E. coli與Shigella spp.在mdh基因相似性高達98%，Shigella bodyii其mdh基因與大腸桿菌之相似性甚至高於與其他Shigella spp.之相似性。 根據動物實驗及細胞組織培養之體外 (in vitro) 試驗，沙門氏菌之病源性與腸毒素 (enterotoxin)有關，但目前為止僅部份血清型沙門氏菌之腸毒素基因被調查。本研究針對沙門氏菌腸毒素stn 基因自行設計PCR引子組，檢測A 至W沙門氏菌共23種不同血清型80株菌株之stn基因之分布，並利用CHO細胞毒性分析佐證毒性蛋白之存在，期能找出菌株之致病性與腸毒素二者之相關性。結果顯示，受試之80株沙門氏菌菌株93.8 %具有腸毒素stn基因，6.2 %菌株不具有stn基因存在，缺少stn基因之菌株為Salmonella oranienburg(SO 24)、S. richmond(SR 03)、S. sandiego(SS 15)、S. tennessee(ST 182)、S. vejle(SV10)等5株；而PCR陰性反應菌株，以細胞毒性分析結果菌株毒性亦明顯減弱，兩者結果一致；而缺乏腸毒素stn基因之Salmonella菌株在動物及食品中致病感染菌株分離率，也鮮少發現此血清種菌株。 大腸桿菌O157及沙門氏菌為食品中重要之食品中毒病源菌，本研究室乃針對大腸桿菌E. coli O157及沙門氏菌自行設計PCR引子組，發展多套式聚合酶鏈鎖反應法(multiplex PCR)，用以同時檢測大腸桿菌E. coli O157及沙門氏菌，並應用於食品兩菌之同時檢測，當檢測目標菌等菌量混合，配合預培養增菌步驟，可檢測原樣品中100 CFU之兩種目標菌；若檢測目標菌混合菌數相差102倍以上時，直接檢測或以GN broth預培養增菌，均無法同時檢測出兩目標菌。若兩目標菌經增殖並配合免疫磁株分離法(Immunomagnetic separation)，即使兩目標菌比例相差105倍也可由多套式聚合酶鏈鎖反應法檢測出來。可解決不等目標菌量於多套式聚合酶鏈鎖反應法無法檢測出全部目標菌之缺點，將上述多套式聚合酶鏈鎖反應法系列應用於食品檢測時，食品成分與非目標之雜菌並不會干擾此多套式聚合酶鏈鎖反應法之檢測。 ; Escherichia coli is an index orgainism for fecal contamination of foods and environments. Infection by pathogenic E. coli strains may cause human diarrhea disease or food poisoning case. On the food safety viewpoint, rapid method for E. coli detection is thus important. On E. coli detection, both the PCR and DNA hybridization methods have been developed. However, differentiation between E. coli and Shigella spp. has always been difficult. Previously, we have developed malate dehydrogenase gene (mdh) based PCR primers for the specific detection of E. coli cells, and found that Shigella spp. could generate false positive results. In this report, we found that by DNA sequencing technique, the mdh gene sequences for Shigella spp. show a sequence homology higher than 98% as compared to those of the E. coli cells. According to the results from in vivo and in vitro tests, Salmonella enterotoxin has been found important to the virulence of Salmonella cells. However, on enterotoxin assay, only part of the serovar of Salmonella strains have been investigated for the presence of enterotoxin (stn) gene. We thus, in this study, designed stn specific primers and used them for the survey of the presence of stn gene in most of the Salmonella strains including 80 Salmonella strains with 23 serovarieties. Furthermore, the CHO cell cytotoxicities for these Salmonella strains were also investigated so that the relationship between stn gene and virulence for Salmonella strains may be elucidated. Results show that for these 80 Salmonella strains, 93.8% show positive PCR results. 6.2% of these strains, ie, strains of Salm. oranienburg(SO 24), Salm. richmond(SR 03), Salm. sandiego(SS 15), Salm. tennessee(ST 182), Salm. vejle(SV 10) show negative PCR results. These five strains, are negative in cytotoxicity test using CHO as target cells. In addition, frequencies for isolation of these strains from infective cause are very low. Since both E. coli O157 and Salmonella spp. are important for food-poisoning cases, we thus also designed a multiplex PCR system which allowed the simultaneous detection of E. coli O157 and Salmonella spp. in foods. On using this multiplex PCR system, it was found that if the target cell number ratio were higher than 102, both target cells could not be detected simultaneously, even a preculture step using GN broth was performed prior to PCR. However, when immunomagnetic separation (IMS) method using equimolar mixture of anti-Salmonella and ant-E.coli O157 immuno-beads was performed prior to PCR, both target cells could be detected even the ratios of these target cells were up 105. Also, as the IMS-PCR method was used for PCR inspection of food samples, food components and the naturally contaminated non-Salmonella microflora could not interfere with the detection. ; 目 次 頁 次 中文摘要………………………………………………………………………….1 英文摘要………………………………………………………………………….2 第一章、研究背景與目的…………………………………………………….4 第二章、文獻整理……………………………………………………….…….7 第一部份、志賀氏菌之簡介…………………………………………………….7 一、志賀氏桿菌之型態及生理特性………………………………………….7 二、志賀氏桿菌之致病原因………………………………………………….7 三、志賀氏菌與食品中毒之關係…………………………………………….8 四、分子生物學生物技術之檢測…………………………………………….8 第二部分、大腸桿菌之簡介……………………………………………………10 一、大腸桿菌……………………………………………………………….10 二、致病性大腸桿菌 (pathogenic E. coli)………………………………….10 三、腸出血性大腸桿菌 (EHEC) ………………………………………….11 (一)流行病學上EHEC的發現………………………………………….11 (二)感染症狀…………………………………………………………….11 (三)傳染媒介…………………………………………………………….12 (四)Escherichia coli O157:H7之生化及生長特性…………………….12 (五)致病因子…………………………………………………………….13 (六)檢測方法…………………………………………………………….14 (1) 選擇性培養………………………………………………………….14 (2) 免疫分析…………………………………………………………….15 (3) 免疫磁株分離法(Immunomagnetic separation; IMS)……………….15 (4) 特異性噬菌體……………………………………………………….16 (5) DNA探針分析法(DNA probe assay)發展………………………….16 (6) 聚合酶鏈鎖反應(Polymerase chain reaction; PCR)及其應用………17 第三部分、沙門氏菌之簡介……………………………………………………18 一、沙門氏菌之型態與生理特性………………………………………….18 二、沙門氏菌與食品中毒之關係………………………………………….19 三、沙門氏菌感染之症狀………………………………………………….19 四、沙門氏菌之致病因子………………………………………………….20 (一)毒素之產生…………………………………………………………20 (二)表面抗原……………………………………………………………21 (三)致病性質體…………………………………………………………21 (四)侵入基因(invasion gene)……………………………………………22 五、沙門氏菌之傳統檢測………………………………………………….22 六、分子生物技術應用於沙門氏菌之檢測……………………………….22 (一)DNA探針分析法……………………………………………………22 (二)免疫分析……………………………………………………………23 (三)免疫磁株分離法(Immunomagnetic separation; IMS)…………….23 (四)聚合鏈鎖反應(Polymerase chain reaction; PCR)與及其應用…….24 表(2-1~2-4)…………………………………………………………………….26 圖(2-1~2-4)…………………………………………………………………….30 第三章、志賀氏菌部份mdh基因序列之定序…………………….….34 壹、前言……………………………………………………………………….34 貳、材料與方法……………………………………………………………….35 一、實驗材料……………………………………………………………….35 二、實驗方法……………………………………………………………….38 (一)PCR引子組之設計…………………………………………38 (二)引子組之合成………………………………………………38 (三)聚合鏈鎖反應(PCR)……………………………………….38 (四)PCR產物的純化……………………………………………38 (五)Gel extraction回收PCR產物………………………………39 (六)PCR產物之定序……………………………………………39 (七)分子選殖……………………………………………………39 1. LB/ampicillin/IPTG/X-Gal plate之製作……………………39 2. DNA片段與定序載體之連接………………………………39 3. 勝任細胞 (Competent cells) 之製作………………………40 4. 轉型作用 (Transformation)…………………………………40 5. 質體之抽取………………………………………………….40 參、結果與討論……………………………………………………………….41 一、E. coli之PCR檢測……………………………………………………41 二、Shigella部分mdh基因序列之定序………………………………….41 三、Shigella定序結果…………………………………………………….42 肆、結論……………………………………………………………………….46 表(3-1~3-2)…………………………………………………………………….48 圖(3-1~3-24)……………………………………………………………………50 第四章、以PCR檢測沙門氏菌腸毒素基因之存在………………….82 壹、前言…………………………………………………………………….82 貳、材料與方法…………………………………………………………….82 一、實驗材料……………………………………………………………….82 二、實驗方法……………………………………………………………….84 (一)PCR引子組之設計……………………………………………….84 (二)引子組之合成………………………………………………………84 (三)聚合酶鏈鎖反應(PCR)…………………………………………….84 (四)PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism)…………………………………………………….85 (五)細胞培養……………………………………………………………85 (六)細胞解凍與保存……………………………………………………85 (七)細胞毒性分析………………………………………………………85 1. 沙門氏菌腸毒素之製備……………………………………….85 2. CHO細胞之毒性分析試驗…………………………………….86 參、結果與討論………………………………………………………………86 一、以PCR檢測沙門氏菌之腸毒素stn基因………………………….86 二、CHO細胞之毒性分析…………………………………………………87 肆、結論…………………………………………………………………….89 表(4-1~4-2)……………………………………………………………………90 圖(4-1~4-7)……………………………………………………………………94 第五章、應用免疫磁株於多套式PCR同時檢測大腸桿菌O157及沙門氏菌……………………….…………………….101 壹、前言……………………………………………………………………101 貳、材料與方法……………………………………………………………102 一、實驗材料………………………………………………………………102 二、實驗方法………………………………………………………………104 (一)聚合酶鏈鎖反應(PCR)………………………………………….104 1. 特異性………………………………………………………….104 2. 靈敏度………………………………………………………….104 (二)樣品之檢測與應用………………………………………………105 1. 乳品之檢測與應用………………………………………………105 (1) 牛乳中生菌數之計算……………………………………….105 (2) 牛乳中大腸桿菌與沙門氏菌之增菌培養………………….105 (三)免疫磁株於樣品檢測之應用……………………………………105 1. 乳品之檢測與應用………………………………………………105 (1) 牛乳中大腸桿菌與沙門氏菌之增菌培養…………………105 (2) 免疫磁株分離法之應用…………………………………….105 2. 牛肉食品之檢測與應用……………………………………….106 (1) 牛肉食品大腸桿菌及沙門氏菌之增菌培養……………….106 (2) 免疫磁株分離法之應用…………………………………….106 參、結果與討論…………………………………………………………….107 一、多套式PCR(Multiplex PCR)可行性之探討…………………………107 二、多套式PCR引子組檢測之靈敏度………………………………….107 三、應用多套式PCR引子同時檢測食品中大腸桿菌O157與沙門氏菌…………………………………………………………………108 四、食品中不同大腸桿菌O157與沙門氏菌之菌量比率對多套式PCR結果之影響………………………………………………….109 五、應用免疫磁株分離法於食品之檢測…………………………………110 (一)免疫磁株分離法之可行性……………………………………….110 (二)免疫磁株分離菌株之效率……………………………………….111 (三)應用免疫磁株於多套式PCR同時檢測大腸桿菌O157與沙門氏菌……………………………………………………………111 (1) 乳品之檢測…………………………………………………….112 (2) 牛肉食品之檢測……………………………………………….112 肆、結論…………………………………………………………………….113 表(5-1~5-4)………………………………………………………………….114 圖(5-1~5-12)…………………………………………………………………118 參考文獻……………………………………………………………………….130