臺灣博碩士論文加值系統

食物中毒是全世界最常見的感染性疾病之一，其中細菌性食物中毒病原具有最多種類也最複雜，因此如何儘快釐清食物中毒病原細菌為重要的議題。傳統方法檢測整體流程曠日廢時，而隨著跨領域檢驗技術研發，能對細菌進行準確且快速的檢測，其中核酸檢測是最具潛力的檢測方式之一。本研究利用高解析度融解曲線(high-resolution melting curve, HRM)法研發對供試食物中毒病原菌(包括Bacillus屬、Staphylococcus屬、Escherichia屬、Salmonella屬、Shigella屬、Pseudomonas屬及Vibrio屬共7屬DNA樣本)快速檢測技術。自GenBank擷取各菌種的核醣體DNA(ribosomal DNA)序列進行多重序列排比(multiple sequence alignment)，在rDNA的不同片段設計引子進行聚合酶鏈鎖反應(polymerase chain reaction)，探討16S rDNA、ITS(internal transcribed spacer)擴增子進行HRM分析，並依據HRM剖面圖(HRM profile)判別種間、亞種菌株間的差異。再進一步以改良的半巢式PCR(hemi-nested PCR, hn-PCR)進行16S rDNA變異區的擴增與HRM分析，並依據HRM剖面圖歸群，結果顯示16S rDNA變異區的hn-PCR，可以大幅提升HRM微分曲線的解析度，進而能輕易分辨至少7種菌株，甚至可解析不同亞種菌株間的差異。此外，由鄰接法(neighbor-joing method)建立的16S rDNA序列為基礎的類源樹(phylogenetic tree)，也驗證類源關係越相近的菌株，在HRM微分曲線剖面圖之波峰的位置及形狀也有越相近的趨勢。本研究並探討其適用範圍，以及未來構築鑑定平台的發展方向。

Foodborne illness is one of the most common infectious diseases worldwide. Among them, food poisoning syndromes caused by bacterial pathogens are known to be very diversified and complicated. Therefore, how to clarify food poisoning pathogens as soon as possible is an important issue. The conventional methods used for the diagnosis of bacterial food poisoning pathogens are very time-consuming. However, with the advance of cross-cutting diagnostic technology, many new tools are available for an accurate and rapid bacterial detection and diagnosis. Among them, nucleic acid-based detection and diagnosis is one of the most promising sectors in in vitro diagnostics. In this study, a high-resolution melting curve method (HRM) was employed for developing the detection and identification methods of the above- mentioned pathogens. Totally, 7 genera of DNA samples, including Bacillus spp., Staphylococcus spp., Escherichia spp., Salmonella spp., Shigella spp., Pseudomonas spp. and Vibrio spp. were used. The ribosomal DNA sequences of each species were retrieved from the GenBank and aligned, the primers for polymerase chain reaction (PCR) was subsequently designed. The HRM profiles of the 16S rDNA and ITS (internal transcribed spacer) amplicons were examined to characterize inter-species and inter-subspecies difference. The amplicons of the 16S rDNA variable regions were obtained lastly with the hn-PCR(hemi-nested PCR) protocol, and resolved then by the HRM analyses. The results have clearly demonstrated that the HRM profiles revealed by the aforementioned approach could differentiate at least 7 species of the 5 different genera. Moreover, it was capable of resolving the difference at the subspecies level. Additionally, the phylogenetic relationship reconstructed using the neighbor-joining method, based on the 16S rDNA sequences of the examined species, was also shown that the closer the phylogenetic relationship, the similar the HRM profile. The potential application and prospect of this study as well as the plausible approach to build an identification and diagnostic platform were addressed finally.

中文摘要(Chinese abstract) i
英文摘要(English abstract) ii
目錄 iv
圖目錄 vii
表目錄 x
壹、 緒論 1
一、 前言 1
二、 研究背景及文獻回顧 2
1. 台灣常見細菌性食物中毒病原 2
2. 選擇適合微生物分子檢測的生物標記 6
3. 快速檢測引發食物中毒病原菌的方法 8
4. 高解析度融解曲線分析法 10
5. 應用於DNA條碼分析上的聚合酶鏈鎖反應 11
三、 研究動機與目的 12
貳、 實驗材料與方法 14
一、 實驗藥品與儀器 14
二、 菌種與DNA樣本來源 15
三、 菌種純化與保存 17
四、 液態懸浮培養 18
五、 菌株基因體DNA萃取 18
六、 引子設計 18
七、 PCR測試條件 19
八、 瓊脂膠體電泳 20
九、 HRM分析 21
參、 實驗結果 22
一、 菌種培養及DNA萃取結果 22
二、 檢測食物中毒病原菌之16S序列 22
1. 利用16S序列之引子對7屬病原菌進行PCR初步檢測HRM曲線 22
三、 檢測食物中毒病原菌之16S-ITS序列 23
1. 利用5'-16S-ITS序列之引子對5屬病原菌進行PCR檢測HRM曲線 23
四、 檢測食物中毒病原菌之ITS序列 24
1. 利用16S-3'-ITS序列之引子對5屬病原菌進行PCR檢測HRM曲線 24
2. 比較5'-16S-ITS序列之引子與16S-3'-ITS序列之引子對5屬病原菌之HRM曲線分析 25
五、 改良食物中毒病原菌16S序列之檢測 26
1. 利用16S序列之引子對5屬病原菌進行16S保守區hemi-nested PCR檢測HRM曲線 26
2. 利用16S序列之引子對5屬病原菌進行16S變異區hemi-nested PCR檢測HRM曲線 27
3. 比較不同濃度16S序列之引子對5屬病原菌進行16S變異區hemi-nested PCR檢測HRM曲線 27
4. 比較16S序列之引子與16S-5'-ITS序列之引子對5屬病原菌進行PCR檢測HRM曲線及16S序列之引子對5屬病原菌進行16S變異區hemi-nested PCR檢測HRM曲線 29
5. 正常化16S序列之引子對5屬食物病原菌進行16S變異區hemi-nested PCR檢測HRM曲線 29
6. 利用16S序列與ITS序列之引子對4種病原菌進行16S變異區hemi-nested與ITS序列duplex PCR初步檢測HRM曲線 30
肆、 討論 32
一、 HRM融解曲線圖之檢測程序 32
二、 PCR擴增序列長度對HRM解析度的影響 33
三、 Nested PCR引子間的競爭關係 35
四、 對可疑菌株及偽陽性條帶進行定序確認 37
五、 探討HRM對混雜菌株的分析能力 39
伍、 結論及未來展望 40
陸、 參考文獻 42
柒、 圖 53
捌、 表 83
玖、 附錄 94
附錄一、實驗流程圖 94
附錄二、利用HRM分析不同PCR擴增子設計流程示意圖 95

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