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研究生:賴威舜
研究生(外文):Wen-shun Lai
論文名稱:質譜技術於臨床微生物檢測之應用
論文名稱(外文):Mass Spectrometry based approaches for the Identification of Microorganisms in Clinical Samples
指導教授:胡安仁
指導教授(外文):Anren Hu
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:133
中文關鍵詞:質譜微生物毛細管電泳液相層析噬菌體
外文關鍵詞:mass spectrometrybacteriacapillary electrophoresisbacteriophageHPLC
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本實驗共可分成兩部分,第一部份以毛細電泳及基質輔助雷射脫附游離質譜
法快速鑑定泌尿道感染病原菌。毛細管電泳(CE)是一種應用廣泛、分析快速、再
現性高的分析儀器,近年來已成為生物分析研究的重要技術之一。第一部分實驗
主要結合毛細管電泳儀與MALDI-TOF 質譜儀來快速鑑定泌尿道感染病原菌,以
往傳統診斷微生物過程需耗費大量時間與金錢,以泌尿道感染診療為例,現今的
尿液檢驗方法,即使由資源完善的實驗室來做分析及樣品處理,仍需費時2~3 天
的時間。然而以我們的方法應用在臨床的尿液檢體中能快速鑑定病原菌,且能使
菌體依然存活。此方法僅需費時30 分鐘。相較於傳統的塗盤培養檢驗方法要快
速許多。鑑定方法首先將臨床的尿液檢體以簡單過濾與離心的方式,除去尿液中
的雜質,將病原菌純化出來,接著使用毛細管電泳技術將尿液中的病原菌分離並
且收集,以偵測樣品產生的遷移時間做初步的判定,收集完的菌體再利用
MALDI-TOF質譜儀依菌體表面蛋白質分佈的指紋峰來做菌種之再確認,確診出是
受到何種細菌的感染。此法我們成功的從臨床檢體中鑑定出五隻病原菌包含了
Escherichia coli、Pseudomonas aeruginosa、Staphylococcus aureus、Staphylococcus
epidermidis、Staphylococcus saprophyticus,這套技術能夠快速鑑定出泌尿道感染
的病原菌,耗時短,未來可將此技術運用於診斷人類其他細菌感染的疾病上。
第二部分以二維液相層析微灑串聯質譜法解析多重抗藥性鮑氏不動桿菌噬
菌體之蛋白質。近年來蛋白體體學的研究中,發展出二維液相層析的新技術,只
需將蛋白質先以酵素消化水解成胜�托寣A便送入二維液相層析串聯質譜儀分析
(2D-LC-MS/MS),此法適用於大量複雜蛋白質體的鑑定。在我們的研究中,運用
IV
此技術來分離噬菌體蛋白質。二維液相層析技術由兩種不同的分離管柱所組成,
第一維為陽離子交換管柱,依胜�扛熊旦q點(PI)不同來進行分離,第二維則是C-18
逆相層析管柱,依胜�氖P管柱間的親和力不同來分離,此法與傳統的二維膠體電
泳分離方法比較快速許多,透過這種技術,能在一天內快速鑑定噬菌體或細菌蛋
白質。
本研究分析的樣品來自臺灣北中南東四家教學醫院,將廢水點入多重抗藥性
之鮑氏不動桿菌,收集受感染的菌株噬菌體,培養後加以純化。噬菌體首先以溶
劑萃取與過濾的方法取出噬菌體蛋白質,然後將蛋白質以胰蛋白�◆簿懦i行消化
水解,最後將消化過的混合胜�成洏峇G維液相層析進行分離。第一維分離管柱為
強陽離子交換管柱,使用十種不同pH 值的緩衝溶液進行沖提,pH 值分別為(2.5、
3.0、3.5、4.0、4.5、5.0、5.5、6.0、7.0、8.0)等10 種,收集十瓶不同pH 值所沖
提的樣品,再各別以C-18 逆相層析管柱進行第二維的分離,樣品經由C-18 管柱
分離後直接送入串聯質譜儀偵測,所得到的結果使用Biowork 生物資訊軟體搜尋
比對,鑑定噬菌體蛋白質。運用二維液相層析質譜法成功的解析出四隻噬菌體,
不論在Online 或Offline 2D-LC-MS/MS 系統上皆能鑑定出10 個左右的蛋白質,
並得到噬菌體豐富的生物資訊,在未來將藉由這些訊息,進一步研究病毒與寄主
間的相互作用與基因轉移的現象。
This experiment can be divided into two parts. First, the rapid identification of
pathogens in urinary tract infections by CE and MALDI-TOF-MS. Capillary
electrophoresis (CE) has become one of the most significant techniques in biological
research, due to the development of a simple method, high separation efficiency, low
sample consumption and short analysis time. These advantages may also be realized
for the analysis of microorganisms. In this study, we have cleaned up the clinical urine
samples by simple filtration and centrifugation, then the pellets were redispersed in
buffer solution. We used the CE to separate and identify pathogens in urinary tract
infections (UTI). This method is rapid and needs only 30 minutes to identify bacteria
in urine specimens; it was much faster than the conventional biological methods (about
2–3 days). In some special cases, we collected the microbial pathogens from CE, made
a careful check by MALDI-TOF mass spectrometry; it was extremely simple and fast
to identify pathogens through protein fingerprinting. We successfully separated and
identified five species including Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus
saprophyticus. Therefore, the work presented with the advent of powerful CE and
MALDI-TOF methods, for an accelerated discovery and diagnosing of diseases in
human.
Secondary, the analysis of bacteriophage proteome of multidrug resistance
Acinetobacter baumannii by 2D-LC-Nanospray-MS/MS. In recent years
multidimensional separation techniques (2D-LC-MS/MS), which the protein mixture
is digested prior to mass spectrometry analysis, was developed for high-throughput
VI
proteome analysis. In this technique, proteins/peptides are separated on the basis of
their iso-electric point (pI) in the first dimension and by their molecular mass in the
second dimension. The first dimension is made up of strong cation exchange (SCX)
and the second dimension with C18 material. By using this method we analyzed the
phage proteins of bacteria within one day, it was faster and almost automates than
conventional method.
In current study, the Acinetobacter baumannii bacteriophage was isolated from
waste water of four hospitals in the north, south, middle and east area of Taiwan. We
collected the multidrugs-resistant Acinetobacter baumannii (MDRAB) bacteria from
waste water, after that cultured and amplified. By using filtration and ultra-centrifuge
to we cleaned up the bacteriophage proteins, and then trypsin used to digest the phage
proteins. The digested phage proteins/peptides were separated using 2-dimension
chromatography. First, we used SCX column to separate the sample as the first
dimension. We eluted the sample with a series of buffers with different pH values (pH
2.5, pH3, pH3.5, pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH7.0 and pH 8.0). We
accumulated the ten eluted samples by different pH buffers. Subsequently, we used
C-18 column to separate the sample as secondary dimension, and directly detected the
spread sample by nanospray mass spectrometry. Finally, we used biowork searching
engine with whole Acinetobacter baumannii bacteriophage BLAST database to
analysis the bacteriophage proteins and predict its function by bioinformatics tool. In
total, the proteins (about ten) of the purified bacteriophage had been identified by
proteomic analysis. Both we successfully got biological information of four
Acinetobacter baumannii bacteriophage strains from off-line and on-line
2D-LC-MS/MS. The bioinformatics of bacteriophage may lead us to new insights into
virus-host interactions and gene transfers in the future.
總目錄
中文摘要 Ⅰ
英文摘要 Ⅲ
目錄 Ⅴ
圖目錄 Ⅸ
表目錄 XI
附錄 XII
第一部份 以毛細電泳及基質輔助雷射脫附游離質譜法
快速鑑定泌尿道感染病原菌
第一章 緒論
一、前言 1
二、研究源起 2
三、文獻回顧 3
四、毛細管電泳簡介 4
五、泌尿道感染 10
六、未來展望 10
第二章 材料與方法
一、實驗藥品 11
二、溶液配製 12
三、儀器 12
四、標準菌體配製 13
VIII
五、毛細管電泳實驗流程 13
六、MALDI-TOF 質譜儀實驗流程 14
第三章 結果與討論
一、緩衝液pH 值對樣品分離影響 15
二、毛細管電泳分析細菌檢體 16
三、傳統方法檢測 20
四、質譜儀技術檢測 21
五、臨床檢體作前處理對於分析上的影響 25
六、臨床檢體作前處理對於回收率的影響 28
七、臨床檢體運用毛細管電泳的分析 30
第四章 結論 33
第五章 參考文獻 35
第二部份 以二維液相層析微灑串聯質譜法解析
多重抗藥性包氏不動桿菌噬菌體之蛋白質
第一章 前言
一、研究源起 39
二、研究目標 40
(1) 鑑定噬菌體蛋白質組成 40
(2) 自動化與非自動化的差異 41
第二章 緒論
IX
一、噬菌體簡介 41
二、多重抗藥性包氏不動桿菌 43
三、文獻回顧 45
第三章 質譜分析技術
一、蛋白質體學研究 47
(1) 電噴灑游離法 ESI 47
(2) 基質輔助雷射脫附游離法 MALDI 48
二、二維液相層析質譜儀介紹 49
第四章 樣品製備與實驗方法
一、樣品配製 50
二、Integrated online-二維液相層析質譜儀實驗流程 52
三、Offline-二維液相層析質譜儀實驗流程 54
四、液相層析質譜儀實驗流程 55
五、MALDI-TOF 質譜儀實驗流程 56
六、生物資訊軟體 57
七、儀器設備 57
八、實驗藥品 59
第五章 結果與討論
一、以Integrated online-二維液相層析質譜儀分析Phage AB1 蛋白質 60
二、以Offline-二維液相層析質譜儀分析Phage AB1 蛋白質 61
三、比較Online 與Offline-二維液相層析質譜儀系統 62
四、以Offline-二維液相層析質譜儀分析不同Strain 噬菌體 63
X
五、以MALDI-TOF 質譜儀解析噬菌體蛋白質組成 82
六、以液相層析質譜儀解析噬菌體蛋白質組成 84
第六章 結論 89
第七章 參考文獻 91
第一部份 以毛細電泳及基質輔助雷射脫附游離質譜法
快速鑑定泌尿道感染病原菌
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