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標題: Tuf 基因序列用於乳酸菌之分子鑑定與定量及其與16S rRNA基因在雙歧桿菌親緣性分析之比較
Use of Tuf gene Sequences for the Qualitative and Quantitative Assay of Lactic Acid Bacteria and Comparison of Tuf with 16S rRNA gene for the Phylogenetic Analysis of Bifidobacterium
作者: 許勝傑
Sheu, Sen-Je
關鍵字: tuf gene
Multiplex PCR
Denaturing gradient gel electrophoresis
Real-time PCR
出版社: 食品暨應用生物科技學系所
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摘要: 具益生菌特性之乳酸菌廣泛使用於發酵食品、乳製品之製造以及食品或飼料添加物。對於消費者或主管機關而言,了解益生菌產品中乳酸菌菌種以及其活菌數是非常重要的。16S rRNA基因一直被認為是真細菌菌種鑑定最佳的標的基因。然而,卻有著相近菌種之16S rDNA 序列間具高度相似性以及基因體中不同operon間可能有不同的序列的問題存在。Tuf 基因為細菌延展因子 (elongation factor Tu) 蛋白之基因,在革蘭氏陽性細菌中只有一套。近年來,常用於設計分子檢測方法之替代基因。因此,本研究試著以tuf 基因為標的基因,開發乳酸桿菌及雙歧桿菌之分子鑑定與分子定量方法。 首先,經由比對基因庫中之乳酸桿菌之tuf基因與recA基因序列,由tuf基因序列設計九組特異性引子組及以recA基因序列設計四組特異性引子組,可以分別檢測L. acidophilus、L. brevis、L. casei group、L. delbrueckii、L. farciminis、L. fermentum、L. jensenii、L. reuteri、Lactobacillus spp.及L. amylovorus、L. pentosus、L. plantarum/L. brevis、L. rhamnosus。另外,以自行設計的引子組直接檢測市售標榜內含乳酸桿菌之乳製產品,結果顯示PCR (Polymerase chain reaction) 之檢測與產品標示相符。因此,所開發之引子組,可應用於乳製品中乳酸桿菌之檢測。 另一方面,針對18株雙歧桿菌部分tuf 及16S rRNA之基因進行定序分析,經比較其序列相似度後發現14株不同菌種之部分 tuf 及16S rRNA基因序列相似度分別為 82.24~99.72% 與 92.33~99.05%,部分 tuf 基因序列有較多之變異性,於雙歧桿菌之鑑別上提供較佳之區分能力。另外,以部分 tuf 基因序列進行分析之親緣演化樹與以部分16S rRNA基因序列分析之結果相似,tuf 基因序列可作為菌種親緣演化分析之工具。根據雙歧桿菌tuf基因序列,可設計13組特異性引子組,分別檢測B. adolescentis、B. animalis、B. bifidum、B. breve、B. cuniculi、B. gallinarum、B. globosum、B. indicum、B. infantis、B. longum、B. minimum 、B. subtile 及Bifidobacterium spp.。針對常使用於乳製品之B. lactis 與B. longum,檢測靈敏度皆可達N×103 CFU/ml (N=1~9) 之菌數。而以自行設計的引子組直接檢測市售標榜內含雙歧桿菌之乳製產品,並配合平板法計數活菌,可用來確認乳製產品之品質。 在多套式PCR (Multiplex PCR) 開發部分,選擇常使用於發酵乳之乳酸菌為開發標的,利用前述 tuf基因序列設計之Laci_tF/ Laci_tR、Lcasg_tF/ Lcasg_tR、Ldel_tF/ Ldel_tR、Blon_tF/ Blon_tR PCR 引子組開發一多套式PCR系統,可分別檢測 L. acidophilus、L. casei group、L. delbrueckii與B. longum,其PCR產物大小分別為397、230、202與161 bp,於牛乳樣品中之檢測靈敏度可達N×103 CFU/ml之菌數。利用此多套式PCR直接檢測市售標榜內含乳酸菌之發酵乳產品,並以文獻發表之16S rDNA 或 16S-23S ITS序列所設計引子組與API50 CHL套組作確認,其檢測結果顯示具有良好之特異性。此多套式PCR可應用於發酵乳產品中,乳酸桿菌與雙歧桿菌之快速檢測。 變性梯度膠體電泳 (Denaturing gradient gel electrophoresis, DGGE) 可以區分相同長度而不同序列之DNA片段,使用 PCR配合DGGE可以分析一混合菌相中之個別菌種。本研究針對 Bifidobacterium 之 tuf 基因序列設計專一性引子,開發一 Bifidobacterium PCR-DGGE 之鑑定系統,對菌種有很高的鑑別能力,可以將16個菌種 (包含2個subspecies)分成13群:B. adolescentis/B. thermophilum、B. indicum、B. subtile、B. boum、B. longum/B. magnum、B. cuniculi、B. minimum、B. breve、B. animalis、B. bifidum、B. lactis/B. gallinarum、B. globosum與B. infantis;若配合吾人所開發tuf gene-based PCR引子組,則可使16個菌種 (包含2個subspecies) 全部被鑑定出來。使用tuf gene-based PCR-DGGE方法,再配合tuf gene-based PCR或定序確認,將可非常實用的應用於檢測樣品中之Bifidobacterium菌種。 另外,以 tuf 基因所設計之L. acidophilus、L. casei group、L. delbrueckii、B. lactis與B. longum特異性引子組,配合ABI 7500 Real-time PCR 系統及SYBR Green I為染劑,進行即時聚合酶鏈反應,結果顯示,以產生之螢光訊號,輔以PCR產物 melting temperature (Tm) 值裂解溫度的偵測,五組引子組皆得到良好的特異性。在添加目標菌之牛乳中,即時聚合酶鏈反應之靈敏度可達102~103 CFU/ml。而在定量分析方面,三市售優酪乳產品在儲存過程中L. acidophilus、L. casei group、L. delbrueckii、B. lactis與B. longum菌數,以MRS-sorbitol agar、MRS agar (pH 5.2)、MRS-vancomycine agar 與BIM-25 agar等選擇性培養基篩選菌落後經PCR確認之菌數與以即時聚合酶鏈反應檢測DNA量,經換算後所得之菌數有相當之一致性。乳酸菌即時聚合酶鏈反應較傳統PCR快速、具較高檢測靈敏度,並在定量上有開發之潛力。
Lactic acid bacteria (LAB) strains with probiotic functions have been used for the processing of fermented food and milk products as well as food and feed supplements. In addition to viability, identity of the LAB species in products is important to consumers and regulatory agencies. 16S rRNA gene has been generally used as target for the identification of eubacteria. However, the identical 16S rDNA sequences of closely related species and the divergent 16S rDNA sequences of a single organism remain problems. The elongation factor Tu gene (tuf), which is present as a single copy in most gram-positive bacteria, has recently been an alternative for designing gene-based methods. In this study, we tried to develop and use of tuf gene-based molecular methods for the qualification and quantification of Lactobacillus and Bifidobacterium. Based on tuf and recA gene sequences retrieved form the GenBank database, 12 species-specific and one genus-specific primer sets were designed for the PCR detection of L. acidophilus, L. brevis, L. casei group, L. delbrueckii, L. farciminis, L. fermentum, L. jensenii, L. reuteri, Lactobacillus spp. and L. amylovorus, L. pentosus, L. plantarum/L. brevis, L. rhamnosus. The specificities of these PCR primer sets were confirmed by assaying Lactobacillus spp. and other bacterial strains. All the targeted Lactobacillus generated PCR products with predicted sizes. Furthermore, the labels of commercial fermented milk products were examined with these PCR primer sets. The results indicated that label of most fermented milk products were correct with Lactobacillus species. Therefore, the specific PCR primers developed could be used to detect Lactobacillus in fermented milk products. On the other hand, partial sequences of the tuf and 16S rRNA gene for 18 Bifidobacterium strains belonging to 14 species were determined. Phylogenetic tree was constructed using neighbor-joining method. The phylogenetic tree based on tuf gene has a profile similar to that determined on the basis of 16S rRNA gene. Sequence alignment for these sequences showed that the similarities among the 14 Bifidobacterium species were 82.24-99.72% for partial tuf genes and 92.33-99.05% for partial 16S rRNA genes, respectively. According to these partial tuf genes, 12 species-specific and one genus-specific primer sets were designed for the PCR detection of B. adolescentis, B. animalis, B. bifidum, B. breve, B. cuniculi, B. gallinarum, B. globosum, B. indicum, B. infantis, B. longum, B. minimum, B. subtile and Bifidobacterium spp.. While Bifidobacterium spp. and other bacterial strains were assayed, these PCR primer sets showed great specificities to targeted Bifidobacterium. Detection limits of species-specific PCR were N×103 CFU per ml (N=1-9) in artificially spiked milk targeted with B. longum and B. lactis, respectively. Bifidobacterial strains in commercial probiotic products could be identified with these tuf gene-based PCR primers and be enumeration by plating count method. Thus, the quality of these porbiotic products could be assured. In addition, tuf gene-based PCR primers specific for the detection of L. acidophilus, L. casei group, L. delbrueckii, and B. longum were combined for the simultaneous detection of these LAB. The specificity of this multiplex PCR was confirmed and the PCR products generated from L. acidophilus, L. delbrueckii, L. casei group, and B. longum were 397, 230, 202, and 161 bp, respectively. The identification limit for each LAB strain with this multiplex PCR method was N× 103 CFU per ml in milk samples. When this multiplex PCR method were used for the simultaneous detection of the LAB in fermented milk products, the LAB species listed on the labels of these products could be identified without the preenrichment step. The results of the multiplex PCR method were further confirmed by PCR assay using primers based on the 16S rDNA or the 16S-23S intergenic spacer region and by biochemical tests using the API 50 CHL kit. In conclusion, this multiplex PCR method could be used for the identification and detection of LAB in commercial fermented milk products. Denaturing gradienet gel electrophoresis (DGGE) is a technique able to differentiate DNA fragnments with sequences diversity. With polymerase chain reaction, DGGE was able to analyze the species in mixed cultures. PCR-DGGE targeting tuf gene was developed and used for species identification. When 16 Bifidobacterium species were assayed with the PCR-DGGE method, all these species could be differentiated to 13 separated patterns, except that the tuf gene sequences from B. adolescentis/B. thermophilum, B. longum/B. magnum and B. lactis/B. gallinarum which migrated the same distance with DGGE gel. Combing with species-specific PCR, all the 16 Bifidobacterium species could be identified. Detection limits of genus-specific PCR-DGGE were N×104 CFU per ml in artificially spiked milk targeted with B. longum and B. lactis, respectively. Furthermore, species-specific PCR and PCR-DGGE were used for the detection of bifidobacterial species in probiotic products. The Bifidobacterium in probiotic products could be faithfully detected by tuf gene-based PCR and PCR-DGGE. Finally, SYBR Green I based Real-time PCR were developed for the detection of L. acidophilus, L. casei group, L. delbrueckii, B. lactis and B. longum using tuf gene-based specific primers. According to the melting temperature (Tm) of the PCR products generated with these primers, the specificity could be confirmed. Using Ct (cycle threshold) and the concentration of bacteria cells, the standard curves of LAB strains were generated and could be used in evaluating the concentration of bacteria. For the quantification of the LAB in fermented products, the enumeration results obtained from selective medium plating and Real-time PCR showed great agreement between two methods. The result suggested that it has potential for developing a culture-independent bacteria enumeration procedure by Real-time PCR.
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