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研究生:陳志遠
研究生(外文):Chen, Chih Yuan
論文名稱:利用熱休克蛋白基因設計Clostridiumtyrobutyricum及Listeriamonocytogenes之PCR引子組及其應用
論文名稱(外文):Development and application of PCR primers for Clostridium tyrobutyricum and Listeria monocytogenes using heat shock protein gene
指導教授:李世傑李世傑引用關係曾浩洋曾浩洋引用關係
指導教授(外文):Shih-Chieh LeeTsen, Hau Yang
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:83
中文關鍵詞: 病原菌 熱休克蛋白基因 特異性 鮮乳 乾酪
外文關鍵詞:pathogensheat shock protein genespecificmilkdry cheese
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  • 被引用被引用:2
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Listeria monocytogenes 廣泛分布自然界的病原菌,而傳染給人類,其為現代食品衛生上極為重要的一株病原菌,也經常出現在生的食品,以及許多熟食或加工食品,尤其低溫殺菌的牛乳、乾酪和冰淇淋、煙燻的魚或肉類製品。另外 Clostridium tyrobutyricum 雖然非病原菌,然其常在乾酪及生乳中會分離出來。因該菌會破壞乾酪及牛乳品質,因此快速檢測是很重要的,因為在傳統檢測方法通常很費力費時。而本研究中建立兩對新穎特異性引子組,分別針對 C. tyrobutyricum 及 L. monocytogenes 進行檢測。而因為 C. tyrobutyricum 之熱休克蛋白未發表,本研究因此先利用 Clostridium 屬內菌株之已發表之熱休克蛋白基因比對,分析並選擇基因序列之保守區段設計引子組,嘗試增幅出 C. tyrobutyricum 之熱休克蛋白基因片段並進行定序確認。之後利用定序結果之熱休克蛋白基因序列,設計引子 CT-1/CT-2,以及 L. monocytogenes 之類熱休克蛋白基因設計引子 Lm1/Lm2,分別針對 C. tyrobutyricum 及 L. monocytogenes 使用 PCR 增幅進行 PCR 引子組特異性檢測。使用引子組 CT-1/CT-2 進行檢測,除了 C. tyrobutyricum 會產生 104 bp 之預期產物外,其他菌株,包含其他 Clostridium 屬菌株,皆不會產生偽陽性之干擾。而由 L. monocytogenes 之類休克蛋白基因引子進行 PCR 增幅,可得到 120 bp 之預期產物外,其他菌株,包含其他 Listeria 屬,皆不會產生偽陽性之干擾。當引子組 CT-1/CT-2 應用於檢測鮮乳樣品中 C. tyrobutyricum 時,在 PCR 增幅前,先進行 24 小時之預培養後,其檢測靈敏度為 N×100 CFU/ml;而應用於檢測乾酪樣品,其靈敏度為 N×100 CFU/g。而引子組 Lm1/Lm2 應用於食品樣品中 L. monocytogenes 檢測,經 12 小時預培養後,鮮乳及乾酪樣品的檢測靈敏度均可達到 N×100 CFU/ml 及 N×100 CFU/g。
本研究亦利用 Real-time PCR 方法,以引子組 CT-1/CT-2 及 Lm1/Lm2 檢測鮮乳及乾酪樣品中之 C. tyrobutyricum 及 L. monocytogenes。當引子組 CT-1/CT-2 應用於檢測鮮乳樣品中 C. tyrobutyricum 時,在 Real-time PCR 增幅前先進行 24 小時之預培養後,其檢測靈敏度為 N×100 CFU/ml;而應用於檢測乾酪樣品,其靈敏度為 N×100 CFU/g。而引子組 Lm1/Lm2 應用於食品樣品中 L. monocytogenes 檢測,經 12 小時預培養後,鮮乳及乾酪樣品的檢測靈敏度可達到 N×100 CFU/ml 及 N×100 CFU/g。
Listeria monocytogenes is one of the common food pathogens. Contamination of the bacteria species on raw foods and cooked or processed foods, including low temperature sterilized milk, cheese and ice cream, smoked fish or meat is common, on the other hand, clostridium tyrobutyricum, which is often isolated from dry cheese and row milk, may spoil the quality of cheese and milk. Since conventional method for detection of these bacteria species is laborious and time consuming, rapid method is very important. In the study, we designed the PCR primers from the conserved region of hsp gene of clostridium spp. and used the primer set for the amplification of hsp gene of C. tyrobutyricum followed by sequencing to obtain the hsp gene sequence of C. tyrobutyricum. Through the alignment and comparison of the hsp gene of C. tyrobutyricum and L. monocytogenes to those genes available in Genebank, primers CT-1/CT-2 and Lm1/Lm2 specific for C. tyrobutyricum and L. monocytogenes were obtained. Using CT-1/CT-2, a PCR product with molecular weight of 104 bp could be obtained. Bacteria speices other than C. tyrobutyricum including other clostridium spp. would not generate any false positive reaction. On the other hand, Lm1/Lm2 allowed the amplification of a 120 bp PCR product. None of other bacteria species including the Listeria spp. would not generate positive reaction. As CT-1/CT-2 was used for PCR detection of target cells in dry cheese, the detection limit was N×100 CFU/g if the target cells were pre enrichment for 24 hrs, anaerobic.
Similarly, the detection limit for milk and dry cheese samples using Lm1/Lm2 was N×100 CFU/ml or g is a 12 hrs pre enrichment step was performed prior to PCR.
The this study, CT-1/CT-2 and Lm1/Lm2 were also used for real-time PCR detection of C. tyrobutyricum and L. monocytogenes. Under such condition, if a 24 hrs or 12 hrs per enrichment steps were performed prior to PCR, a detection limit of N×100 CFU/ml or g for milk or dry cheese could be obtained for both, detection cases.
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.....................................................................................................vii
目錄....................................................................................................viii
圖目錄..................................................................................................xi
表目錄................................................................................................xiii

1.文獻回顧.............................................................................................1
1.1 Clostridium…...............................................................................1
1.1.1 Clostridium 分類................................................................1
1.1.2 Clostridium 之特性............................................................1
1.1.3 Clostridium 形態................................................................2
1.1.4臨床上重要致病性 Clostridium菌株...............................2
1.1.5 Clostridium tyrobutyricum...................................................3
1.1.6 C. tyrobutyricum 之快速檢測............................................3
1.2 Listeria monocytogenes................................................................4
1.2.1李斯特菌之分類.................................................................5
1.2.2 L. monocytogenes 之一般特性...........................................5
1.2.3血清型分類與致病決定位(Virulence determinants)..........6
1.2.4 L. monocytogenes 之快速檢驗方法……………….......6
1.3 熱休克蛋白的介紹....................................................................8
1.4 即時 PCR (Real-Time PCR) 介紹.........................................10
1.5 實驗目的..................................................................................13
2.材料方法..........................................................................................15
2.1 實驗材料..................................................................................15
2.1.1 菌株...............................................................................15
2.1.2 培養基...........................................................................15
2.1.3 藥品...............................................................................15
2.1.4 緩衝液及試劑...............................................................16
2.1.5 儀器...............................................................................17
2.2 實驗方法......................................................................................18
2.2.1 熱休克蛋白基因分析及篩選…....................................18
2.2.2 PCR 引子組設計...........................................................19
2.2.3 PCR引子組及寡核苷酸引子之合成............................20
2.3 聚合酶鏈鎖反應 (PCR)..........................................................20
2.3.1 DNA製備.......................................................................20
2.3.2 C. tyrobutyricum 之 DnaK 基因定序..........................21
2.3.3引子組CT-1/CT-2 及 Lm1/Lm2 之 PCR 特異性
試驗................................................................................22
2.4 CT-1/CT-2 及 Lm1/Lm2 於食品檢驗之應用….....................22
2.4.1 CT-1/CT-2 鮮乳樣品的檢驗應用…..............................22
2.4.1.1直接檢測…........................................................22
2.4.1.2 增菌培養….......................................................23
2.4.2 CT-1/CT-2 乾酪樣品的檢驗應用….............................24
2.4.3 Lm1/Lm2 鮮乳樣品的檢驗應用..................................24
2.4.3.1 直接檢驗..........................................................24
2.4.3.2 增菌培養..........................................................24
2.4.4 Lm1/Lm2 乾酪樣品的檢驗應用..................................24
2.5 Real-Time PCR (即時聚合酶鏈反應)......................................25
3.結果與討論......................................................................................26
3.1 C. tyrobutyriucm 及L. monocytogenes之序列比對...............26
3.2 聚合酶鏈鎖反應 (PCR)..........................................................27
3.3 食品檢測之應用…......................................................................29
3.3.1 傳統 PCR 檢測靈敏度...................................................29
3.3.2 即時 PCR 檢測靈敏度................................................30
3.3.3 Real-Time PCR標準曲線之建立..................................31
4.結論...................................................................................................33
參考文獻…..........................................................................................71
附錄......................................................................................................79


圖目錄

圖 1. CTO1/CTO2 增殖出 C. tyrobutyricum DnaK 基因結果.......36
圖 2. Clostridium 屬熱休克蛋白基因 DnaK 序列比對結果與特
異性引子組設計位置................................................................37
圖 3. Listeria spp. 類熱休克蛋白基因 HtpG-like 基因部份片段
序列比對結果與特異性引子組設計位置…............................39
圖 4. CT-1/CT-2 檢測 Clostridium spp. 結果..................................40
圖 5. CT-1/CT-2 檢測其它非目標菌之結果….................................41
圖 6. 引子組 Lm1/Lm2 檢測 L.monocytogenes 之結果...............42
圖 7. 引子組 Lm1/Lm2 檢測 Listeria spp.結果...........................43
圖 8. 引子組 Lm1/Lm2 檢測其它非目標菌結果...........................44
圖 9. 引子組 CT-1/CT-2 直接檢測牛奶內之C. tyrobutyricum
傳統 PCR結果........................................................................45
圖10. 引子組 CT-1/CT-2 直接檢測乾酪內之C. tyrobutyricum
傳統 PCR結果........................................................................46
圖11. 引子組 CT-1/CT-2 增菌培養條件下牛奶內之
C. tyrobutyricum 傳統 PCR結果...........................................47
圖12. 引子組 CT-1/CT-2 增菌培養條件下乾酪內之
C. tyrobutyricum 傳統 PCR結果...........................................48
圖13. 引子組 Lm1/Lm2 直接檢測牛奶內 L.monocytogenes 傳統PCR結果..................................................................................49
圖14. 引子組 Lm1/Lm2 增菌培養條件下牛奶內
L. monocytogenes 傳統 PCR結果….....................................50
圖 15. 以引子組 Lm1/Lm2 直接檢測乾酪內 L.monocytogenes
之傳統 PCR 結果.................................................................51
圖 16. 以引子組 Lm1/Lm2 增菌培養條件下檢測乾酪內 L.monocytogenes 之傳統 PCR 結果...................................52
圖 17. 引子組 CT-1/CT-2 直接檢測鮮乳內C. tyrobutyricum 之 Real-time PCR 圖譜..............................................................53
圖 18. 引子組 CT-1/CT-2 直接檢測乾酪內C. Tyrobutyricum 之 Real-time PCR 圖譜..............................................................54
圖 19. 以引子組 Lm1/Lm2 直接檢測鮮奶內L. monocytogenes 之Real-time PCR Tm 值圖譜....................................................55
圖 20. 以引子組 Lm1/Lm2 直接檢測乾酪內 L.monocytogenes 之Real-time PCR Tm 值圖譜....................................................56
圖 21. 以引子組 Lm1/Lm2 增菌培養條件下檢測牛奶內L.monocytogenes 之Real-time PCR Tm 值圖譜.................57
圖 22. 以引子組 Lm1/Lm2 增菌培養條件下檢測乾酪內 L.monocytogenes 之Real-time PCR Tm 值圖譜.................58
圖 23. 以引子組 Lm1/Lm2 直接檢測鮮乳樣品中之 L.
monocytogenes 所得標準曲線..............................................59
圖 24. 以引子組 Lm1/Lm2 直接檢測乾酪樣品中之 L.
monocytogenes 所得標準曲線..............................................60


表目錄

表1. E.coli O157與常見病原菌及本研究目標菌株之熱休克蛋白
家族基因之比對相似度結果...................................................61
表2. 前人研究發表,針對 L. monocytogenes 之 Hly 基因與 C. tyrobutyricum之16s-23s rRNA檢測之 PCR 引子組...........62
表3. 本研究所使用的 PCR 引子組................................................63
表4. 本研究所使用之PCR反應條件..............................................64
表5. 即時 PCR 與傳統 PCR 方法於食品樣品系統中檢測 C. tyrobutyriucm 或 L. monocytogenes 之靈敏度......................65
表6. 李斯特菌屬各菌種之血清型分類…………………………....66
表7. 本實驗所使用之 Clostridium spp. 與 Listeiria spp. 之標準
菌株及PCR結果......................................................................67
表8. 本實驗所使用之非目標菌株及 PCR 結果............................68
表9. 本實驗所使用之非目標菌株及 PCR 結果............................69
表10.本實驗所使用之非目標菌株及 PCR 結果............................70
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