臺灣博碩士論文加值系統

摘要
常久以來細菌培養是鑑定咽部細菌最重要的方法，但細菌培養有其先天上的限制。傳統上咽部細菌鑑定以培養的方式，再加上其生化特性、形狀、染色等來辨識其種類，但對於無法培養之細菌則無法以此方法來鑑定。本實驗嘗試以非傳統之細菌培養方式研究咽炎病人咽部菌種，以直接將細菌細胞打碎的方式，並抽取其中之去氧核醣核酸，以聚合酵素連鎖反應方法直接將其16S rDNA序列放大後，在變性梯度凝膠中行電泳分析。並選殖部分16S rDNA序列，定序後以細菌之16S rDNA片段依其演化之序列，配合基因庫之比對來檢定咽部之菌種。

由單一選殖菌落之16S rDNA於變性梯度凝膠中比對而鑑定的菌種可分辨出其中可分為Actinomyces、Abiotrophia、Actinobacillus、Anaerosphaera、Neisseria、Porphyromonas、Prevotella、Streptococcus和Veillonella。而由選殖16S rDNA所得菌種，主要是Actinomyces、Abiotrophia、Actinobacillus、Anaerosphaera、Granulicatella、Haemophilus、Neisseria、Porphyromonas、Prevotella、Streptococcus和Veillonella。由選殖16S rDNA所得菌種和變性梯度凝膠可辨識菌種比較，變性梯度凝膠可辨識菌種佔選殖16S rDNA所得菌種42.8%。於16S rDNA選殖所得菌種及變性梯度凝膠可辨識菌種中，並無發現於細菌培養中最容易培養出之細菌Streptococcus viridans、Staphylococcus areus 及Staphylococcus epidermis。

由變性梯度凝膠中可發現人類咽部細菌為混合性的細菌生長狀態，可分為多個色帶，急性咽炎病人咽部細菌之16S rDNA序列於變性梯度凝膠中呈現不同之菌相，老年人之咽部細菌種類及菌量與年青人比較有明顯增加，男女之間則無可辨識之差異，於所有變性梯度凝膠菌相中並無發現單一明顯之菌種為優勢菌種。正常人咽部細菌和急性咽炎病人16S rDNA 於變性梯度凝膠中呈現不同之菌相，由變性梯度凝膠可辨識菌種中可發現Streptococcus明顯的增加。

Abstract
For a long time, cultures are the most important method to differentiate bacterial strains in clinical laboratories. But there are limitations on cultures. Traditionally we analyze the pharynx bacterial strains by their cultures, biochemical traits, stains and shapes. This experiment tries to investigate the bacteria communities of pharynx in a non-traditional way. The bacteria are crashed in the first. Polymerase chain reactions are performed thereafter. The 16S rDNA sequences are amplified and electrophoreses in degenerative gadient gel. Some of the 16S rDNA sequences are cloned into vectors, then sequenced. By phylogenic analysis with the database of 16S rDNA sequences, the bacteria strains are determined by sequences.

By the comparison of single cloned colony of 16S rDNA in degenerative gradient gel, the following bacterial strains are recognized: Actinomyces, Abiotrophia, Actinobacillus, Anaerosphaera, Neisseria, Porphyromonas, Prevotella, Streptococcus and Veillonella. By phylogenic analysis of the cloned sequences, the following bacterial strains are recognized: Actinomyces, Abiotrophia, Actinobacillus, Anaerosphaera, Granulicatella, Haemophilus, Neisseria, Porphyromonas, Prevotella, Streptococcus and Veillonella. Only 42.8% of the phylogenic putative strains are found in degenerative gradient gel analysis. The most found strains Streptococcus viridans, Staphylococcus aureus and Staphylococcus epidermis. in cultures are not discovered in both phylogenic putative strains and degenerative gel analysis.

In the degenerative gradient gel electrophoresis, we can see patients with acute pharyngitis have different patterns of gel bands. Multiple bands indicate that the pharynx bacterial community is a mixed flora community. There are more bands and denser bands in aged than young are. No differences between male and female are discovered. No single dominant band is found in any of the pharyngitis degenerative gel. There is a distinct difference between the degenerative gel of pharyngitis patient and normal. In the degenerative gel of pharyngitis, an obvious increase in Streptococcus is noticed.

目錄…………………………………………………………………………I
圖目錄……………………………………………………………………III
表目錄…………………………………………………………….………IV
附錄目錄………………………………………………………….……….V
名詞縮寫對照表…………………………………………………….……VI
中文摘要……………………………………………………….…….…VII
英文摘要……………………………………………………….……..…IX
壹、緒論……………………………………………………………………1
一、細菌分類方法………………………………………………………1
二、人類咽部細菌研究…………………………………………………4
貳、方法與材料
一、細菌採樣……………………………………………………………6
二、細菌DNA備製……………………………………………………….6
三、聚合酵素連鎖反應 …………………………………………………6
四、變性梯度凝膠電泳…………………………………………………7
五、細菌16S rDNA選殖…………………………………………………10
六、16S rDNA片段定序…………………………………………………14
七、基因庫之比對 ……………………………………………….……14
八、化學藥品與儀器設備……………………………. …………….….16
參、結果
一、聚合酵素連鎖反應放大16S rDNA…………………………………18
二、急性咽炎病患變性梯度凝膠之比較………………………………18
三、基因庫比對…………………………………………….………….19
四、16S rDNA變性梯度凝膠位置圖……………………………………24
五、Human oral bacterium C73之菌種確定…………………………24
六、樹狀圖…………………………………………………………….…24
七、咽炎病患與正常人咽部變性梯度凝膠比較………………………25
肆、討論
一、咽部細菌DNA抽取方法……………………………………………26
二、急性咽炎病患咽部細菌變性梯度凝膠分析………………………26
三、正常人與急性咽炎病患變性梯度凝膠分析………………………27
四、生物防治法於急性咽炎之應用……………………………………28

伍、參考文獻…………………………………………………………..29
圖……………………………………………………………………….36
表……………………………………………..…………….…………45
附錄…………………………………………..…………….…………a

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