2006-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/653028摘要：發展快速檢測及篩選乳酸菌之方法有其必要性及重要性，主要原因益生菌 (probiotics) 對人體健康的正面效益已經被許多研究證實，且其中絕大部分皆為乳酸菌，自生物體或環境中篩選新的乳酸菌株，在業界及學界已是重要的研究課題。且乳酸菌已經廣泛的應用在乳製品上，乳酸菌的機能性取決於菌株的種類，因此檢測及鑑定乳製品中所使用之原生菌菌株對於乳製品品質的控制、機能性及發酵過程的監控非常重要。過去幾年本研究室試著利用傳統法及專一性分子探針鑑別乳酸菌發現，傳統法檢測菌種之方式對於未知樣品之菌相，需單獨針對特定菌種進行培養及進一步之試驗，極為耗時且鑑別力有限。分子生物技術進行菌種鑑別、分類與檢測，尚未有一高感度的方法可快速、準確的達成此項工作。本研究擬發展乳酸菌寡核&#33527;酸微陣列(oligonucleotide microarray)法進行常見之乳酸菌群鑑別，並將此新一代分生技術實際應用於動物產品中乳酸菌之篩檢及鑑定包括牛腸胃道及糞中篩選乳酸菌和乳製品之檢驗上。期能開發出快速、正確、敏感度高且可同時區別多種菌種之分生檢測法。 本計畫擬分兩部分，三年先後進行，第一部分為選擇乳品中常見之三十株乳酸菌，根據GeneBank所獲取之序列設計寡核&#33527;酸探針 (oligoprobes)，以點樣系統之微陣列構築一寡核&#33527;酸微陣列 (oligonucleotide microarray)，再以含有標的物 (target) 之DNA樣品進行雜合反應，利用雜合與否之呈色訊號結果分析該晶片之檢測效果，並藉由修正探針設計、微陣列點製條件與樣本DNA萃取等方式，以確保該晶片之正確性及標準化。第二部分則是評估乳酸菌晶片之敏感度與實際應用價值。將第一部分試驗中所開發之乳酸菌微陣列晶片實際應用於牛腸胃道及糞中篩選乳酸菌和乳製品之檢驗上，並與變性膠體電泳法做比較，評估各優劣點，期能建立一個快速又精確的乳酸菌種鑑定方法，並可在複雜菌相之樣本中，同時鑑別檢驗多種乳酸菌。且藉此方法自牛腸胃道<br> Abstract: Lactic acid bacteria are widely employed as starter cultures in the dairy industry. A reliable identification and high-resolution typing of probiotics strains is essential in nutritional sciences and fundamental food research. Detecting and identifying various species of lactic acid bacteria with rapid method is often important for quality control of dairy products and monitoring fermentation processes. The rapid identification of strains belonging to closely related species is still difficult for both phenotypic methods and genotypic methods. The aim of this study will develop a fast and sensitive microarray method, which is able to detect more than 30 lactic acid bacteria. This project will last for three years. In the first and second years, we will design oligonucleotide probes and prepare oligonucleotide-microarray slides for identifying lactic acid bacteria including Lactobacillus spp., Bifidobacterium spp., Lactococcus spp., Streptococcus spp. and Leuconostoc spp. In the last year, this oligonucleotide-microarray will be used for the detection and identification of bovine fecal samples and commercial dairy products. Furthermore, additional denaturing gradient gel electrophoresis will also be applied to compare with microarray method.乳酸菌寡核&#33527酸微陣列Lactic acid bacteriaoligonucleotide microarray