臺灣博碩士論文加值系統

沙門氏菌為全球常見之人畜共通傳染病之一。因沙門氏菌所引起之食物中毒，造成嚴重之嘔吐及下痢等症狀，甚至引發其他併發症而導致死亡。在畜牧場，沙門氏菌造成嚴重之經濟損失主要病原之一。因此，快速且準確之診斷沙門氏菌顯得格外重要。由於沙門氏菌之血清型種類繁多，根據ISO 6579:2002分離沙門氏菌之檢測標準方法，檢驗流程耗時約6天，且操作步驟繁瑣。因此，本研究擬藉由表面增強拉曼光譜(Surface enhanced spectroscopy)技術，建立一快速且準確之檢測方法，達到偵測及區分沙門氏菌之目的。本次試驗選用之8種不同血清型沙門氏菌 (S. choleraesuis、S. enteritidis、S. typhi、S. typhimurium、S. dublin、S. newport、S. paratyphi A、S. paratyphi B)，以表面增強拉曼光譜檢測方法與傳統的分子生物學及細菌培養方法，比較檢測沙門氏菌血清型別之特異性及靈敏度。由本研究結果得知，以表面增強拉曼光譜波數(wavenumber) 500~1600 cm-1位置，可見不同的血清型之沙門氏菌株，不同血清型在波數位置558 cm-1、726~735 cm-1、950 cm-1、1043~1051 cm-1、1272~1293 cm-1具有獨特的特徵波峰。依不同血清型沙門氏菌特徵波峰的相似程度，可將這8種沙門氏菌歸類為3組：S. paratyphi A及S. paratyphi B；S. enteritidis、S. typhi、S. typhimurium及S. newport；S. choleraesuis 及S. dublin。選擇取不同血清型的特徵波峰作為變數依據，輔以主成份統計方法分析，可有效提升本方法之特異性，可將這8種血清型之沙門氏菌由歸類為3組提升到至少可區分6個血清型別。此外，在鑑定沙門氏菌所需的時間，不計算預增菌培養步驟所需時間，可縮短至1小時完成。本研究證實，表面增強拉曼光譜檢測方法應用於偵測沙門氏菌，其操作方法簡單、有效地縮短檢測時間且具檢測專一性的特性。如以不同血清型沙門氏菌之特徵波峰，建立更完整之沙門氏菌拉曼光譜參考資料庫，未來適合應用於食品加工廠或畜產品加工廠之品質管制、畜牧場沙氏桿菌環境監測，沙氏桿菌監測等，具在特定檢測領域具有取代傳統檢測方法之潛力。

Salmonella spp. is one of the common zoonosis in the worldwide. However, food poison caused by Salmonella spp., causes severe vomiting, diarrhea and other symptoms, and lead to other complications leading to death. In livestock ranches, it is one of the main reasons of serious economic losses. Therefore, to diagnose rapidly and accurately Salmonella become particularly important. There are many kind of serotypes of Salmonella spp. and the classification are very complicated. According to ISO 6579:2002 standards, detection methods need to identify of Salmonella isolates, it often takes a lot of examination time. In this study, we would like to develop a rapid and accurate detection method surface enhanced raman spectroscopy, to detect and identify different serotypes of Salmonella. The results of this study, surface enhanced raman spectroscopy (SERS) spectra of the eight Salmonella serotypes, namely S. choleraesuis, S. enteritidis, S. typhi, S. 到B, several wavenumbers including 558 cm-1, 726-735 cm-1,950 cm-1 , 1043-1051 cm-1, and 1272-1293 cm-1 were observed. According to resemblances between characteristic peaks, Salmonella samples can be seperated to 3 groups: S. paratyphi A and S. paratyphi B; S. enteritidis, S. typhi, S. typhimurium and S. Newport; S. choleraesuis and S. dublin. Additional analytic process followed the principal component analysis (PCA) method, the specificity of SERS can be improved to achieve the purpose of distinguishing at least about six Salmonella serotypes. In addition, the detection time for distinguishing Salmonella serotypes can be shortened to 1 hour after the pre-incubation culture.We demonstrated that SERS is a simplely, specificity and effectively method for detecting Salmonella serotypes. After developing SERS wavenumber reference databank of Salmonella serotypes, which can be applied to quality control for food processing plants, meat processing factories, environmental monitoring of Salmonella serotypes. In specific target detection, SERS has the potential to replace traditional detection methods.

中文摘要 i
Abstract ii
表目次 vi
圖目次 vii
第一章、緒論 1
1.1 前言 1
1.2 研究動機 1
1.3 實驗設計 1
第二章、文獻回顧 3
2.1 引發消化道疾病之沙門氏菌症事件回顧 3
2.2 消化道型沙門氏菌 3
2.3 拉曼散射(Raman scattering)介紹 4
2.4 表面增強拉曼光譜原理(Surface enhanced Raman spectroscopy) 4
2.5 表面增強拉曼光譜檢測法發展沿革 4
2.6 表面增強拉曼光譜在細菌偵測上之應用 5
2.7 沙門氏菌多重基因之引用及應用 5
第三章、材料與方法 6
3.1 生物材料及來源 6
3.1.1 菌種活化及保存 6
3.1.2 測試樣本製備 6
3.1.3 沙門氏菌血清型分析─多重聚合酶連鎖反應法 7
3.1.4 沙門氏菌血清型分析─選擇性培養基法 9
3.1.5 聚合酶鏈連鎖反應之檢測靈敏度 10
3.1.6 沙門氏菌測試濃度 11
3.2 表面增強拉曼光譜測定 11
3.2.1 表面增強拉曼活性基板(SERS-chip) 11
3.2.2 表面增強拉曼光譜儀偵測條件及結果分析 11
第四章、結果 12
4.1 沙門氏菌分型及增菌 12
4.1.1 多重聚合酶鏈連鎖反應檢測不同血清型之沙門氏菌 12
4.1.2 選擇性培養基鑑別不同血清型之沙門氏菌 12
4.1.3 利用invA 基因獲得PCR檢測極限 13
4.1.4 沙門氏菌濃度計數 13
4.2 表面增強拉曼光譜技術於沙門氏菌血清鑑別測定 13
4.2.1 不同血清型之沙門氏菌特徵波峰決定 13
4.2.2 特徵波峰再現性 14
4.2.3 拉曼光譜技術偵測靈敏度分析 14
4.2.4 主成份分析結果(Principle component analysis) 15
第五章、討論 16
第六章、結論 19
第七章、參考文獻 62
附錄一、菌瓶開封與活化 66
附錄二、PCR產物之序列比對結果 67
附錄三、作者簡介 83

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