大腸桿菌之毛細管電泳快速檢測技術

Capillary electrophoresis for rapid detection of Escherichia coli

吳淑芬(Shu-Fen Wu)

指導教授 ： 張煥宗

國立臺灣大學/理學院/化學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10050

摘要

環境中之微生物影響人類健康甚鉅，其中大腸桿菌（Escherichia coli, E. coli）為監控水質最理想之指標微生物（indicator microorganism），因此建立有效及快速的大腸桿菌檢測方法，才能即時進行適當的防護措施。本研究係利用毛細管電泳暨雷射誘導螢光偵測於電滲流（electroosomotic flow, EOF）存在下，以含1.7% 聚環氧乙烯（poly(ethylene oxide), PEO）（Mw 4M g/mol）之Tris-borate緩衝溶液進行線上濃縮及分離大體積（約1.0 μL）之蛋白質樣品及細菌溶解產物（lysate）。為解決蛋白質吸附的問題，在樣品中加入0.01% PEO（Mw 300k g/mol），同時於樣品區帶前注入一小段（1.3 cm）0.2%之十二烷基硫酸鈉（sodium dodecyl sulfate, SDS）溶液塞，以提高濃縮與分離效率。將此技術應用於大腸桿菌溶解產物之分析，成功地從複雜基質中標定出其特徵蛋白質訊號峰。由於高濃縮效率提升偵測靈敏度，實際注入毛細管內的菌數只需約3 × 105個。此外，本方法樣品需求量低且不需繁複的前處理步驟，分析環境水樣中之大腸桿菌，可在一個工作天內得到結果，為大腸桿菌檢測提供一個快速、簡單及經濟的方法。

關鍵字

大腸桿菌 ； 指標微生物 ； 毛細管電泳暨雷射誘導螢光偵測 ； 線上濃縮 ； 細菌溶解產物

並列摘要

The specific detection of Escherichia coli (E. coli) is essential for water quality control because its presence points directly to the presence of enteric disease-causing bacterial. We developed a rapid, easy and economical method for the analysis of bacterial lysates by capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF). Addition of 0.01% poly(ethylene oxide) (PEO) (Mw 300k g/mol) to the sample matrix and applied a 0.2% sodium dodecyl sulfate (SDS) plug (1.3 cm) before injecting large-volume (ca. 1.0 μL) sample zone can improve stacking and separation efficiencies. After injection of the large-volume samples, the proteins migrate against the electroosmotic flow (EOF) and enter 1.7% PEO (Mw 4M g/mol) zone; this process causes them to slow down and stack at the boundary between the PEO and sample zones. As a result, the characteristic peaks of E. coli lysate were identified by injecting as few as 3 × 105 cells. Owing to growth rapidly and predominance in fecal contaminated water, we could detect and identify culturable E. coli cells in 50 mL of pond water in campus within one working day.

並列關鍵字

Escherichia coli ； indicator microorganism ； capillary electrophoresis coupled with laser-induced fluorescence ； on-line concentration ； bacterial lysates

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