Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51092
標題: Application of Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis in the Detection and Identification of Lactobacillus, Staphylococcus and Alicyclobacillus Species
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
作者: Chen, Shin-Chih
陳信志
關鍵字: DGGE;DGGE;tuf基因;益生菌;葡萄球菌;環脂酸芽孢桿菌;檢測;鑑定;不需培養;tuf gene;probiotic;Staphylococcus;Alicyclobacillus;detection;identification;culture-independent
出版社: 食品暨應用生物科技學系
摘要: 
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.
URI: http://hdl.handle.net/11455/51092
Appears in Collections:食品暨應用生物科技學系

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