Using molecular targeted methods for the identification of Bifidobactrium from market products.

本試驗以兩項分子標定技術針對市售七種產品之雙叉乳桿菌進行檢測，分別為：種間專一性引子（species-specific primers）及變性梯度膠體電泳（denatural gradient gel electrophoresis）。首先自食品工業研究所生物資源保存中心購得十株標準菌種（Bifidobacterium angμlatum、B. animalis、B. bifidum、B. breve、B. catenμlatum、B. infantis、B. longum、B. minimum、B. subtile與B. thermophilum）。種間專一性引子部分，針對雙叉乳桿菌16S-23S rRNA gene區域設計專一性引子，先藉由分子生物資料庫GeneBank取得目標基因序列後，進行多條序列比對，選擇適當的變異區做為參考點，設計各種雙叉乳桿菌菌株所屬專一性的引子，收集添加雙叉乳桿菌之市售產品並利用純雙叉乳桿菌菌株作為參考值，以傳統鑑別法諸如革蘭氏染色法、型態觀察、RAP ID快速鑑定套組及觸酶試驗等測試，初步判讀其菌種後，再藉由自行設計引子組進行試驗。變性梯度膠體電泳則是利用前人所設計之共通引子組，針對標準菌種進行PCR-DGGE針對各菌種進行試驗以作為標準，引子某端加上GC clamp，令PCR增幅出的雙股DNA一端帶有40~50個GC核酸序列，選擇增幅之序列範圍中，各菌種之鹼基組成必須略有差異，令其於變性梯度膠體中泳動時，因鍵結力不同以達分離目的。 結果顯示本研究所使用兩項分子標定鑑別技術，種間專一性引子及變性梯度膠體電泳於產品A、B及C，皆能夠順利鑑別出商品所標示菌種，此三項產品有一共同特色即為添加菌種皆為單一雙叉乳桿菌菌種酸凝酪，且其商品型式為酸凝酪產品，其中微生物菌相較為單純。而產品D亦是酸凝酪但因標示不明，兩項技術檢驗結果並無對應菌種，其添加菌種身分未明。而在保健菌粉產品E、F及G中，俱添加兩種雙叉乳桿菌及數十種其餘益生菌，其混合微生物菌相複雜，種間專一性引子法之鑑定結果除了商品E中未檢測出B. longum之外，其餘結果均與商品標誌吻合；反觀變性梯度膠體電泳方面，對於混合菌相之檢驗，其結果與商品標示呈現較大的差異。Bifidobacteria are used increasingly and widely in market products in recent years. Confusing occurred due to false declarations and uncritical selection of strains. Detecting and identifying various species of bifidobacteria with a rapid method are often important for quality control of market products. However, the rapid identification of strains of closely related species is still a difficult task. Thus, in this paper, a rapid and reliable gene-targeted species-specific polymerase chain reaction (PCR) technique based on a two-step process was established to identify bifidobacteria in dairy products. The first step was PCR assay for genus Bifidobacterium with genus specific primers followed by the second step，which identified the species level with species-specific primer mixtures. Twenty specific primer sets were developed for each of the Bifidobacterium species including B. angulatum, B. bifidum, B. infantis, B. longum, B. animalis, B. breve, B. catenμlatum, B. minimum, B. subtile and B. thermophilum, and the sets were designed from nucleotide sequences of the 16-23S rRNA gene region. Additional polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and phenotype methods including Gram stain, catalase test and RAPID ANA kit system were applied to confirm the results. The results indicated that all primer sets amplified their targets in the DNA mixture as expected and identified their corresponding target species. Furthermore, the application of gene-targeted species-specific PCR to identify several commercial products demonstrated that four different Bifidobacterium spp. (B. bifidum, B. breve, B. infantis and B. longum) were identified and confirmed by DGGE. Compared with phenotype methods, using the 16-23S rRNA gene-targeted species-specific PCR technique is a simple, rapid and reliable method for identification of bifidobacteria in market products.目 次 頁次 表次.............................................. Ⅳ 圖次.............................................. Ⅴ 壹、中文摘要...................................... 1 貳、緒言.......................................... 3 參、文獻檢討...................................... 4 一、益生菌定義................................. 4 二、益生菌之種類及需具備特性................. 4 三、益生菌促進人體健康之機能................. 5 (一)抑制病原菌.............................. 5 (二)降低膽固醇含量.......................... 6 (三)改善乳糖不耐症.......................... 6 (四)預防和治療下痢.......................... 7 (五)抗癌性................................... 7 (六)改善營養價值............................ 7 四、益生菌菌種鑑別............................ 8 (一)表現型分類法............................ 8 (二)培養基法................................. 9 (三)分子生物鑑定法.......................... 11 五、聚合酶鏈鎖反應............................ 21 (一)引子設計通則............................ 21 (二)PCR反應通則............................ 22 肆、材料與方法................................... 24 試驗一、以種間專一性引子（species-specific primer）鑑別市售產品之雙叉乳桿菌.......................................24 一、材料........................................ 24 二、試藥及培養基............................... 25 三、實驗儀器及器材............................ 27 四、方法........................................ 27 (一)純化DNA................................ 27 (二)菌株初步確認............................ 29 (三)以屬別專一性之引子篩選產品中雙叉乳桿菌及PCR................................. 30 (四)敏感度試驗.............................. 31 (五)種間專一性引子之設計及PCR................ 31 試驗二、以變性梯度膠體電泳（Denatural Gradient Gel Electrophoresis, DGGE）鑑別市售產品之雙叉乳桿菌........................................ 37 一、材料........................................ 37 二、試藥及培養基............................... 37 三、實驗儀器及器材............................ 37 四、方法........................................ 38 伍、結果與討論................................... 43 一、傳統方法................................... 43 二、分子標定法................................. 43 (一)屬別專一性引子之檢測................... 43 (二)靈敏度試驗.............................. 45 (三)種間專一性引子.......................... 48 (四)變性梯度膠體電泳....................... 71 陸、結論.......................................... 87 柒、參考文獻...................................... 89 捌、英文摘要...................................... 96 玖、作者小傳...................................... 98 表 次 頁次 表一、菌種名稱及菌種中心之編號..................... 26 表二、NCBI之菌種序列編號.......................... 34 表三、種間專一性引子之條件......................... 35 表四、各引子組之鏈合溫度........................... 36 表五、DGGE引子組之條件........................... 41 表六、DGGE 凝膠成分............................... 42 表七、10%過硫酸銨成分............................. 42 圖 次 頁次 圖一、變性梯度膠體電泳分析之流程................... 20 圖二、種間專一性引子實驗流程....................... 33 圖三、PCR-DGGE實驗流程............................. 40 圖四、a.革蘭氏染色法 b.RAPID ANA之結果............ 44 圖五、以雙叉乳桿菌屬專一性引子組P0及Lm3，針對標準菌株進行PCR之結果.......................... 46 圖六、以雙叉乳桿菌屬專一性引子組P0及Lm3，針對樣品進行PCR之結果.............................. 47 圖七、以專一性引子組Bang，針對標準菌株進行PCR之結果.......................................... 51 圖八、以專一性引子組Bbif，針對標準菌株進行PCR之結果.......................................... 52 圖九、以專一性引子組Bin，針對標準菌株進行PCR之結果.......................................... 53 圖十、以專一性引子組Blon，針對標準菌株進行PCR之結果.......................................... 54 圖十一、以專一性引子組Bani，針對標準菌株進行PCR之結果...................................... 55 圖十二、以專一性引子組Bbre，針對標準菌株進行PCR之結果...................................... 56 圖十三、以專一性引子組Bcat，針對標準菌株進行PCR之結果...................................... 5