臺灣博碩士論文加值系統

齲齒和牙周病兩種口腔疾病與人類口腔微生物群組的關聯已經有多年深入的研究。而口腔及周邊疾病的有效疾病管理，則需要一個快速有力的檢測、鑑定口腔微生物的平台。本研究之目的在發展一個基礎於HRM(High-resolution Melting)技術的快速、簡單可靠、經濟有效之口腔細菌篩選技術。HRM技術運用PCR放大片段的熔解曲線解析供試DNA樣本的序列細微變異。實驗為71位受試者口腔篩選的62株細菌，以螢光染劑LC Green+標記的16S及 23S擴增DNA片段，利用HR-1儀器即時偵測放大片段熔解過程中不同溫度的螢光值、由熔解曲線進而解析其序列變異。使用16S通用引子對﹝8F/1492R(l)﹞進行62株菌株的HRM分析，配合其中24株代表性菌株的DNA序列分析所提供之分子分類學資訊，可將供試菌株區分為屬階層的11類群。為提高專一性，使用筆者設計的16S引子對(178165/178163)和23S引子對(1782352/1782332)再對上述24株菌株進行第二次分析，結果顯示16S組比23S組更能有效分辨菌株；而由其分子歸群所得結論雖然與前述試驗相同，但是由再設計的引子對所得較短放大片段之HRM圖譜一般鑑別度較高。最後模擬田野取得菌種樣品組成複雜的情況，使用混合已知物種的DNA進行PCR-HRM分析結果顯示此方法分辨樣品中的菌種組成之成效並不理想。

The association of two oral diseases, dental caries and periodontal disease, and microbial communities present in human oral cavities has been well-documented for decades. However, a fast and powerful detection, identification platform for assessing oral microbes is needed for an effective disease management of above-mentioned diseases. The aim of this research is to develop a rapid, simple yet reliable, cost-effective screening method using the HRM(High-Resolution Melting) technology. The HRM technique, basing on detecting small differences in PCR melting curves, resolves small sequence variations in DNA samples examined. Totally 62 bacterial isolates from 71 subjects were studied. The level of fluorescence versus temperature of the fluorescent dye LC Green+-labeled 16S and 23S amplicons were real-timely monitored using the HR-1 instrument to generate the melting curves for subsequent assessment of sequence variations. Eleven groups, providing genus-level identification, could be classified by using the HRM profiles of the 16S fragments amplified from 62 isolates with the 16S universal primers 8F/1492R(1), together with the molecular taxonomic information of the 24 representative isolates. To increase the sensitivity, the HRM profiles of 24 isolates were further examined with the shorter DNA fragments amplified with primer sets 178165/178163(for 16S amplicon) and 1782352/1782332(for 23S amplicon), respectively. The results revealed that the HRM profiles of the 16S amplicons were more powerful in clustering examined isolates than those of 23S. Regarding the molecular typing, although the conclusion remained unchanged, their HRM profiles were in general more characteristic than the previous ones. Lastly, to mimick the mixed composition of microbes normally found in field samples, different known DNA samples were mixed and analyzed with the PCR-HRM protocol, the results indicated that the current protocol was incapable of effectively distinguishing the component species in all cases.

壹、緒論...1
一、前言...1
二、文獻回顧...2
三、研究動機與目的...8
貳、實驗材料與方法...10
一、實驗藥品與儀器...10
二、菌種與DNA樣品來源...11
三、液態和固態培養基製備...11
四、菌種固態培養...11
五、菌種液態培養...12
六、菌種純化與保存...12
七、菌體DNA萃取...12
八、引子對設計...13
九、聚合酶連鎖反應(polymerase chain reaction, PCR)測試條件...14
十、瓊脂膠體電泳...14
十一、高解析度熔解曲線分析(high resolution melting, HRM)分析...14
參、結果...16
一、使用LA平板篩選觀察口腔菌株...16
二、使用單一組引子對進行未鑑定口腔菌株分析...16
1. 4位受試者12菌株單引子對放大16S部分片段之電泳、HRM及DNA定序分析...16
2. 67位受試者50菌株單引子對放大16S部分片段之電泳、HRM及DNA定序分析...18
三、使用單一組引子對進行未鑑定口腔菌株二次分析...21
1. 4位受試者12菌株單引子對放大16S部分片段之電泳、HRM及DNA定序分析...21
2. 4位受試者12菌株單引子對放大23S部分片段之電泳及HRM分析...22
3. 67位受試者12菌株單引子對放大16S部分片段之電泳、HRM及DNA定序分析...23
4. 67位受試者12菌株單引子對放大23S部分片段之電泳及HRM分析...25
5. 67位受試者2菌株單引子對放大16S及23S部分片段電泳、HRM及DNA定序分析...26
四、使用單一組引子對進行混合不同菌株DNA之HRM分析...27
肆、討論...30
一、16S通用引子對放大片段-HRM和DNA定序...30
二、16S和23S筆者設計引子對放大片段-HRM和DNA定序...30
三、單一引子對(16S)擴增放大混合樣品...33
四、引子對設計...34
五、HRM圖形的序列和溫度差異...35
六、總結...36
圖次...38
表次...56
參考文獻...111
附錄...115

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