臺灣博碩士論文加值系統

世界衛生組織估計1990-2010年發生約20億食物中毒案例，導致89-140萬人的死亡；在臺灣細菌性食物中毒為食物中毒主因，因此如何快速偵測、鑑定食物中毒病原細菌為重要的公衛課題。傳統分類法依賴培養特性、生化特徵等，本研究則評估多種生物標誌、最適化高解析度融解曲線(high-resolution melting； HRM)分析技術，希望能開發低成本、快速而靈敏之檢測、鑑定實驗室純培養樣本、食品中毒事件現場樣本的技術平台。
使用EF-G(elongation factor G)、EF-Tu(elongation factor thermo unstable)、rpoB (beta subunit of RNA polymerase)、sodA(superoxide dismutase A)及FtsZ(filamenting temperature-sensitive mutant Z)等5種生物標誌19個引子，以傳統PCR或半巢式PCR(hn-PCR)擴增5屬7種食物中毒病原細菌(Staphylococcus epidermidis、Staphylococcus aureus subsp. aureus、Escherichia coli、Shigella sonnei、Salmonella enterica subsp. arizonae、Salmonella enterica subsp. enterica、Bacillus cereus)之標誌序列，PCR擴增子以瓊脂膠體電泳分離和LCGreen plus標記的擴增子以HRM分析。融解曲線圖(-dF/dT)比瓊脂膠體電泳更能解析各生物標誌樣本間的分子差異。
能選用為分辨菌株的生物標誌物種互異。藉由傳統PCR擴增子的融解曲線圖，EF-G標誌能用於分辨B. cereus；sodA標誌能用於分辨B. cereus和S. enterica；rpoB標誌能用於辨認五物種(B. cereus、S. enterica、S. aureus、S. epidermidis、E. coli)；EF-Tu和FtsZ標誌則能用於區辨所有供試物種。藉由半巢式PCR擴增子的融解曲線圖，EF-Tu(tsh)標誌能用於分辨B. cereus、E. coli、S. epidermidis；兩種rpoB標誌(rpoB-ba、rpoB-sta.a)能用於專一性辨認B. cereus或S. aureus；FtsZ標誌(FtsZ-bs)標誌則能用於專一性區辨S. epidermidis。
評估食品中毒事件現場，運用HRM技術快速而靈敏檢測多種病原菌共存的情況，自細菌DNA模板混雜(不同物種、不同比例)液，以傳統PCR和以hn-PCR擴增多種標誌序列，各種擴增子的融解曲線圖均難分辨其組成菌種。

The World Health Organization had estimated that approximately two billion foodborne illness outbreaks from 1990 to 2010 caused 0.89 to 1.4 million deaths worldwide. In Taiwan, the main cause of foodborne diseases is resulted from ingestion of foodstuffs contaminated with bacteria and, therefore, how to rapidly detect and identify food-posioning bacteria is an important public health problem. Bacteria are classified traditionally using cultural characteristics and biochemical features, among others. This study, aiming to develop a technical platform providing a low cost, rapid yet sensitive method for detecting and identifying the lab pure samples and the on-site mixed samples, has evaluated multiple biomarkers and optimized the detection protocol based on the HRM (high-resolution melting) technique.
Totally 19 primers were used to amplify sequences of five biomarkers, namely, EF-G (elongation factor G), EF-Tu (elongation factor thermo unstable), rpoB (beta subunit of RNA polymerase), sodA (superoxide dismutase A) and FtsZ (filamenting temperature-sensitive mutant Z), from seven species (Staphylococcus epidermidis, Staphylococcus aureus subsp. aureus, Escherichia coli, Shigella sonnei, Salmonella enterica subsp. arizonae, Salmonella enterica subsp. enterica, and Bacillus cereus) using traditional PCR and hn-PCR(hemi-nested PCR). The PCR amplicons were separated by agarose gel electrophoresis (AGE), and the LCGreen plus labeled amplicons were analyzed by the HRM techniques. For every biomarker, the melting curve (-dF/dT) was more powerful in resolving the molecular difference among species examined than the AGE method.
The biomarkers could be selected for effective detection varied among bacterial species. Basing on the melting curves of PCR amplicons resulting in amplifications of traditional PCR, biomarker EF-G could be used to specifically detect Bacillus strain. sodA could be used to separate two species (B. cereus and S. enteric). rpoB could be employed to differentiate five species including B. cereus、S. enteric,S. aureus,S. epidermidis and E. coli. Lastly, EF-Tu and FtsZ could be used for differentiation of all examined species. Furthermore, the melting curves of EF-Tu(tsh) amplicons obtained from the hn-PCR protocol could be employed to distinguish B. cereus,S. epidermidis and E. coli. The melting curves of rpoB-ba and rpoB-sta.a amplicons could be used to specifically identify B. cereus and S. aureus, respectively, while those of FtsZ-bs amplicons could be use to specifically detect S. aureus.
To evaluate the potential of HRM technique in on-site detection for rapidly and sensitively screening multiple targets at the same time, the amplicons of various biomarkers resulting in amplifications of traditional PCR and hn-PCR from the DNA mixture solutions (comprising of different DNA targets and different proportion of component DNA templates) were used for subsequent HRM analysis. For every available amplicons it was impossible to identify the component species form the resulting melting curves using the current protocol.

中文摘要(Chinese abstract) i
英文摘要(English abstract) iii
目錄 v
圖目錄 viii
表目錄 x
壹、 緒論 1
一、 常見之食物中毒細菌 2
1.1沙門氏桿菌 2
1.2腸炎弧菌 3
1.3金黃色葡萄球菌 3
1.4肉毒桿菌 4
1.5大腸桿菌 5
1.6仙人掌桿菌 6
1.7志賀氏桿菌 6
二、抗藥性細菌 8
2.1金黃色葡萄球菌 9
2.2大腸桿菌 12
三、食物中毒病原菌的分子檢測 13
3.1檢測用生物標誌 14
3.2半巢式聚合酶鏈鎖反應 17
3.3高解析度融解曲線分析法 17
四、研究動機與目的 19
貳、實驗材料與方法 21
實驗藥品與儀器 21
菌種DNA樣本來源 21
液態懸浮培養 21
菌株基因體DNA萃取 22
引子對設計 23
聚合酶連鎖反應 23
瓊脂膠體電泳 24
高解析度融解曲線分析法 24
參、實驗結果 26
一、螢光染劑加入方式比較 26
二、不同條件進行螢光曲線分析 26
三、檢測食物中毒病原菌 28
1. 利用EF-G序列之引子對5屬病原菌進行HRM分析 28
2. 利用EF-Tu序列之引子對5屬病原菌進行HRM分析 29
3. 利用sodA序列之引子對5屬病原菌進行HRM分析 30
4. 利用rpoB序列之引子對5屬病原菌進行HRM分析 31
5. 利用FtsZ序列之引子對5屬病原菌進行HRM分析 32
6. 不同來源大腸桿菌之比較與HRM分析 33
7. 食物中毒菌DNA混合樣本各目標序列之HRM分析 34
肆、討論 37
一、HRM前處理對融解曲線之影響 37
二、探討相似菌株間HRM分析與鑑定的能力 38
三、混雜菌株與各引子間的競爭關係 42
伍、結論及未來展望 45
陸、 參考文獻 47
柒、 圖 64
捌、 表 105

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