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研究生:陳信志
研究生(外文):Shin-Chih Chen
論文名稱:應用聚合酶連鎖反應-變性梯度膠體電泳法檢測與鑑定乳酸桿菌、葡萄球菌與環脂酸芽孢桿菌
論文名稱(外文):Application of Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis in the Detection and Identification of Lactobacillus, Staphylococcus and Alicyclobacillus Species
指導教授:黃文哲黃文哲引用關係
指導教授(外文):Wen-Zhe Hwang
學位類別:博士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系
學門:醫藥衛生學門
學類:營養學類
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:162
中文關鍵詞:DGGEtuf基因益生菌葡萄球菌環脂酸芽孢桿菌檢測鑑定不需培養
外文關鍵詞:DGGEtuf geneprobioticStaphylococcusAlicyclobacillusdetectionidentificationculture-independent
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變性梯度膠體電泳(Denaturing gradient gel electrophoresis, DGGE)可以區分相同長度而不同序列之DNA片段,使用 PCR(Polymerase chain reaction)配合DGGE可以分析一混合菌相中之個別菌種。
tuf基因為細菌延展因子(elongation factor Tu)蛋白之基因,為一housekeeping基因,tuf基因在革蘭氏陽性細菌中只有一套,而且在某些菌屬之不同菌種間已證明具有比16S rDNA更高的序列差異,因此本研究嘗試以tuf基因為分子標的基因,由其中設計屬專一性(genus-specific)或群組專一性(group-specific)的引子組,進行PCR-DGGE分析。實驗分別設計Lactobacillus、Staphylococcus與Alicyclobacillus之專一性PCR-DGGE,並應用於混合菌種或產品之檢測。
第二章由tuf基因中設計Lactobacillus專一性(Lactobacillus-specific)引子組LbtufF/LbtufR,應用於PCR以檢測Lactobacillus菌株,實驗室所有的Lactobacillus菌種皆有一大約140-b.p.之PCR產物產生,Lactobacillus以外的菌種皆無反應;PCR反應偵測L. acidophilus及L. casei之靈敏度皆為104 CFU/mL。應用此引子組於PCR-DGGE,可以將15個菌種分成13群,相較於使用16S rDNA PCR-DGGE只能將15個菌種分成8群,有明顯較高的解析能力。實驗並分析市售益生菌產品Lactobacillus菌種標示之正確性,結果顯示乳製品之檢測結果大都符合標示,但是膠囊粉末及碇劑類產品卻有80%(4/5)有標示錯誤的情形。
第三章對11個常見的葡萄球菌菌種進行PCR-DGGE分析,結果可以區分為10種不同的圖譜。另外對9株金黃色葡萄球菌菌株、2株沃氏葡萄球菌、2株頭葡萄球菌、2株表皮葡萄球菌以及2株腐生葡萄球菌進行分析,結果指出同種之菌株皆產生相同的圖譜。PCR與PCR-DGGE之靈敏度皆可達到103 CFU/mL。隨機混合數種菌種並添加至牛乳後再進行測試,結果可以偵測出所有添加入牛乳之菌種,其中受測的菌種中包括了具有產毒素潛力之菌種。
第四章由16S rDNA中設計一組引子Ali732F/Ali882R,用以檢測Alicyclobacillus spp.,序列分析顯示此引子組符合目前發表之所有19個Alicyclobacillus菌種的序列,使用實驗室所有之標準菌株與分離珠進行PCR測試,所有的菌株都產生一個大約150-b. p.大小的產物,其他菌屬皆無產物產生;PCR對菌體之靈敏度為102 CFU/mL,而且蘋果汁樣品並不影響靈敏度。實驗另外設計一組引子組GCAli16SF/684R,進行PCR反應得到一大約300-b. p.大小之產物,應用於DGGE分析9個Alicyclobacillus菌種,產生8種不同的圖譜,其中A. acidocaldarius與A. tolerans之片段非常接近而無法區分,但是仍可應用切膠定序的方法進一步區分。
Denaturing gradienet gel electrophoresis (DGGE) is a technique able to differentiate DNA fragnments with sequences diversity. With polymerase chain reaction (PCR), DGGE was able to analyze the species in mixed cultures.
tuf gene is responsible for the synthesis of bacterial elongation factor Tu. The gene is a housekeeping gene and exists only one copy in Gram positive bacteria. It had been proven to possess more diversity than 16S rDNA between species in several genera. As a result, We tried to design genus-specific (or group-specific) primers from tuf gene sequences and practically evaluated in PCR-DGGE analysis of genus Lactobacillus, Staphylococcus and Alicyclobacillus, respectively.
A set of Lactobacillus-specific primers LbtufF/LbtufR was designed in chapter two. Applied in PCR, all Lactobacillus species generated an amplicon about 140-b. p. while genus other than Lactobacillus resulted no products. PCR sensitivity of L. acidophilus and L. casei was 104 CFU/mL, respectively. PCR-DGGE of 15 Lactobacillus species resulted in 13 patterns. Comparison to 16S V2-V3 PCR-DGGE, which differentiated 15 species to 8 patterns, the developed PCR-DGGE obtained a much high resolution. The label accuracies of probiotic products in Taiwan were evaluated. Results indicated label of most dairy products were correct, but 80% (4/5) of tested capsule, tablet and powder products were found to be mislabeled.
In chapter three, eleven common Staphylococcus species were analyzed by PCR-DGGE and resulted in 10 patterns. PCR-DGGE analysis of 9 stains of S. aureus, 2 strains of S. warneri, 2 strains of S. capitis, 2 strains of S. epidermidis and 2 strains of S. saprophyticus revealed that the DGGE patterns were very conserved between strains in a species. When mixed cultures of different Staphylococcus species in milk were sujected to DNA extraction and PCR-DGGE, the resulted patterns faithfully corresponded to the species of the mixed cultures, including those of potential to secret enterotoxins.
A set of Alicyclobacills-specific primers Ali732F/Ali882R was designed from 16S rDNA in chapter four. Sequence analysis indicated the primers matched the sequences of all present 17 Alicyclobacillus species. PCR of the type strains and isolates preserved in our laboratory revealed that each strain generated an amplicon about 150-b. p.. Strains of other genus generated no products. Seneitivity of the PCR were evaluated to be102 CFU/mL, and components in apple juice didn’t interfere the sensitivity. A genus-specific primer set GCAli16SF/684R was also designed in this study for the application in PCR-DGGE analysis. PCR-DGGE analysis of 9 species revealed 8 patterns. Fragments of A. acidocaldarius and A. tolerans were too close to be distinguished. However, sequencing of the amplicons would be suitable to identify the two speices.
頁次 I
圖次 V
表次 VII
中文摘要 VIII
Abstract X
第一章 概論 1
第一節 乳酸桿菌 1
一、乳酸菌之起源與分類 1
(一) 起源 1
(二) 分類 1
二、乳酸菌之特性 3
三、益生菌之介紹 4
(一) 發展歷史 4
(二) 益生菌之條件 4
(三) 益生菌之功能 5
四、發酵乳 8
(一) 發酵乳之歷史 8
(二) 發酵乳之分類 8
五、Lactobacillus之介紹 10
(一) Lactobacillus之特性 10
(二) Lactobacillus之命名、分類與演進 11
(三) 分子標的基因 11
第二節 葡萄球菌 13
一、葡萄球菌之特性 13
(一) 凝固酶(coagulase) 13
(二) 熱穩定性核酸酶(thermonuclease) 14
(三) 腸毒素(Staphylococcal enterotoxins) 15
(四) 中毒休克症候群毒素 1(Toxic shock syndrome toxin-1, TSST 1) 16
(五) 細胞毒素 16
(六) 殺白血球素(Leukocidin) 17
(七) 脫皮毒素(Exfoliative toxins) 17
二、葡萄球菌之分類 17
三、葡萄球菌之重要性 18
(一)醫學上之重要性 18
(二) 食品上之重要性 21
四、葡萄球菌之檢測 23
(一) 傳統檢測 23
(二) 商業套組 25
(三) 分子生物學方法(Genotypic methods) 26
第三節 環脂酸芽孢桿菌 28
一、環脂酸芽孢桿菌之起源與分類 28
二、環脂酸芽孢桿菌之特性 29
三、環脂酸芽孢桿菌孢子與菌體之耐熱性 30
四、環脂酸芽孢桿菌對果汁工業之威脅 30
五、環脂酸芽孢桿菌產生之不良氣味 31
六、環脂酸芽孢桿菌對食品安全之影響 32
七、環脂酸芽孢桿菌之分離與鑑定 32
(一) 篩選性培養基 (Selective media) 32
(二) 分子方法 33
第四節DGGE 35
一、DGGE之發展歷史 35
二、DGGE之操作方法 35
(一) 變性梯度 35
(二) GC clamp 35
三、DGGE之應用 35
(一) 微生物菌相分析 36
(二) 分類或鑑定菌種 36
(三) 分析菌群的消長變化 36
(四) 培養後分析培養基上之主要菌相 36
四、DGGE之使用缺點 36
第二章 應用聚合酶連鎖反應-變性梯度膠體電泳(PCR-DGGE)於鑑定優酪乳及含乳酸菌產品中之混合乳桿菌群 46
一、前言 46
二、實驗材料 49
(一) 儀器 49
(二) 軟體 50
(三) 藥品 50
(四) 菌株 51
(五) 市售益生菌產品 51
三、實驗方法 52
(一) PCR引子組設計 52
(二) 染色體DNA之製備 52
(三) PCR反應之條件 53
(四) 變性梯度膠體電泳 54
(五) DGGE膠片中PCR產物之回收 55
(六) 篩選性培養計數 55
四 結果與討論 55
(一) 分析tuf基因序列 56
(二) 以16S rDNA V2-V3區域為標的基因進行PCR-DGGE 56
(三) 使用引子組GCLbtufF/LbtufR進行PCR-DGGE 57
(四) 引子組GCLbtufF/LbtufR PCR之專一性及靈敏度 57
(五) 市售樣品中乳酸菌之篩選計數 58
(六) 市售樣品之PCR-DGGE分析 59
(七) 市售商品標示之正確性 63
五、結論 64
第三章 應用聚合酶連鎖反應-變性梯度膠體電泳(PCR-DGGE)於鑑定葡萄球菌之混合菌種 85
一、前言 85
二、實驗材料 86
(一) 儀器 86
(二) 藥品 87
(三) 菌株 87
三、實驗方法 87
(一) 快速萃取DNA 87
(二) Staphylococcus-specific PCR 88
(三) DGGE 89
四、結果與討論 89
(一) 使用Staphylococcus-specific引子組之PCR反應 89
(二) DGGE 89
(三) 同一菌種進行PCR-DGGE之保守性 90
(四) 檢測混合菌種 91
(五) 檢測牛乳中之葡萄球菌 91
(六) PCR-DGGE之靈敏度測試 91
五、結論 92
第四章 設計環脂酸芽孢桿菌之專一性引子並應用聚合酶連鎖反應-變性梯度膠體電泳(PCR-DGGE)以檢測多種環脂酸芽孢桿菌種 102
一、前言 102
二、材料與藥品 104
(一) 儀器 104
(二) 藥品 105
(三) 培養基 106
(四) 軟體 106
(五) 菌株 106
(六) 土壤樣品 107
三、實驗方法 107
(一) 篩菌方法 107
(二) Genomic DNA之快速萃取 107
(三) 16S V1-V2區域之PCR-DGGE進行分群 108
(四) 分離株之鑑定 108
(五) 由16S rDNA設計專一性之引子Ali732F/Ali882R 109
(六) Ali732F/Ali882R (Alicyclobacillus-specific) PCR反應 109
(七) 環脂酸芽孢桿菌部分tuf基因序列之選殖與序列分析 110
(八) Alicyclobacillus-specific引子GCAliF/ Ali684R之設計 112
(九) GCAli16SF/ Ali684R PCR-DGGE分析 113
四、結果與討論 113
(一) 由土壤篩選Alicyclobacillus菌株 113
(二) 分離株之分群與鑑定 113
(三) Alicyclobacillus-specific引子組Ali732F/Ali882R之序列評估 114
(四) Ali732F/Ali884R PCR特異性 115
(五) Ali732F/Ali884R PCR靈敏度 115
(六) 部分tuf基因之序列分析 115
(七) GCAli16SF/ Ali684R PCR 116
(八) GCAli16SF/ Ali684R PCR-DGGE 116
五、結論 116
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