藉由變性梯度膠體電泳及寡核苷酸微陣列技術 鑑定乳酸菌之研究

Study on identification of lactic acid bacteria by denaturing gradient gel electrophoresis and oligonucleotide microarray

陳希嘉(Hsi-Chia Chen)

指導教授 ： 陳明汝

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.00289

摘要

本研究嘗試利用聚合酶連鎖反應-變性梯度膠體電泳（PCR-DGGE）與寡核苷酸微陣列（oligonucleotide microarray），兩項分子生物技術建立不同之乳酸菌鑑定平台，測試其分析單一純菌或複合菌相之檢驗能力，期望提高鑑菌結果之正確性與減化檢驗工作之流程。
在PCR-DGGE鑑菌平台建立第一步，即篩選四組引子對於目標乳酸菌群之區隔能力，選擇GC338f/518r引子所增幅之16S rDNA為最佳解析基因區段，使用於克弗爾粒（kefir grains）中之未知乳酸菌之測定與市售含乳酸菌產品之檢測。搭配培養方式分析由不同培養條件下純化之單一菌落，得知MRS培養基結合好氣條件培養可使最多不同種類之乳酸菌於此條件下生長，避免其他不適當培養下之菌群低估，同時配合定序方法與傳統鑑定菌種方式，確知PCR-DGGE鑑定乳酸菌之正確性與應用性。搭配培養方法可確認四不同菌種Lactobcillus kefiranofaciens、Lb. kefiri、Leuconostoc mesenteroides、Lactococcus lactis存在於克弗爾粒中；在不需培養方法中僅Lb. kefiranofaciens、Lb. kefiri或Lc. lactis可被檢出，另外亦檢出E. coli與Pseudomonas spp.之污染菌存在於樣品中。使用PCR-DGGE配合不需培養方法，應用於市售乳酸菌產品之菌種檢測，酸凝酪產品中可穩定測得Str. thermophilus，其次為Lb. delbrueckii subsp. bulgaricus，此二者皆為酸凝酪發酵之常見菌種；乳酸菌飲品檢出之乳酸菌種類常較商品標示者多，最易檢出之菌種為Lb. acidophilus，其次為Lb. casei；凍乾菌粉類產品檢出之菌種則常少於標示者。PCR-DGGE於複雜菌相樣品之檢測效力仍受限於菌數多寡與菌體存活率。
寡核苷酸晶片之設計，首先透過序列資料庫Ribosomal Database Project II取得目標菌群之16S rRNA基因序列，分別利用ARB與PRIMROSE兩探針設計軟體找尋適當之乳酸菌特異性寡核苷酸探針，並藉由安捷倫（Agilent）探針合成技術製作為晶片實體，16S rDNA樣品需進行95℃加熱5小時之前處理，造成1500 bp長度之核酸片段至20∼150 bp，搭配ULS標定法標定目標核酸後，與晶片雜合之訊號強度穩定且極高，可供作為檢驗標準化流程。由5株目標菌（Lb. acidophilus、Lc. lactis、Str. thermophilus、Leu. mesenteroides及Bif. animalis）與1株非目標菌（B. cereus）之純菌樣品，與2混菌樣品（目標菌Lb. acidophilus混合非目標菌B. cereus與5株目標菌同時混合）分別進行雜合反應，依八組晶片表現差異分析探針之有效性，經GeneSpring GX軟體篩選探針後，在31組普遍性探針中，共有24組達預期效果可表現正控制組探針特性，僅有1組完全不具顯著訊號；於3573個乳酸菌探針中，已確認其中11 % 會與非目標菌產生偽陽性訊號，此外，種別探計實際檢測效果較屬別探針為佳，種別探針中表現true-positive訊號者，以Lb. acidophilus、Str. thermophilus與Bif. animalis探針設計效果最佳，可達50 %以上，Lc. lactis探針則僅約8 % 為有效探針，Leu. mesenteroides探針之效果最弱，僅1 % 可表現顯著訊號。非目標菌存在於檢測樣品或是多目標菌混合，並不會干擾有效探針之檢測效力，目標DNA量由0.5

關鍵字

變性梯度膠體電泳 ； 寡核苷酸微陣列 ； 乳酸菌 ； 鑑定

並列摘要

Lactic acid bacteria (LAB) are widely employed as starter cultures in the dairy industry and supplemented in different commercial products. 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. However, identification of lactic acid bacteria in the complex bacterial communities is still difficult for both phenotypic methods and genotypic methods. The aim of this study focused on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and oligonucleotide microarray method to develop a rapid, reproducible and easy to handle molecular tool for identification of lactic acid bacteria in dairy products.
For PCR-DGGE, selection of primers is the key to analyze highly conserved genetic profiles among LAB. Four primer sets, targeted for 20 reference LAB strains, were evaluated and primer set GC338f/518r was selected due to its highly discriminating power at species level. After selection of primer sets, LAB in kefir grains and commercial probiotic products were assessed using PCR-DGGE by a culture-independent and a culture-dependent way, and were further confirmed by DNA sequencing techniques. Kefir grains results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains, Lactobacillus kefiranofaciens, Lb. kefiri, Leuconostoc mesenteroides and Lactococcus lactis from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains. The DGGE profiles for commercial probiotic products demonstrated that the samples contained most lactic acid bacteria that specified on the product label except of the lyophilized mixed starter powder. This may be due to a concentration below the detection limit or due to the fact that none of these bacteria were present in the probiotic product.
For microarray method, oligonucleotide probes targeting variable regions of the 16S rRNA gene were designed and tested for the identification of LAB. The strategy involved designing possible oligomer probes, using two software (ARB and PRIMROSE), for each target at species and genus level. All candidate 25-mer probes were poly(T)-tailed to reach an overall length of 60 oligonucleotides. Microarrays were manufactured by in situ synthesis (Agilent Technologies). Eight samples, including 5 pure LAB (Lb. acidophilus, Lc. lactis, Str. thermophilus, Leu. mesenteroides and Bif. animalis), 1 non-targeted stain (B. cereus) and 2 mixed strain, were then assessed. Results indicated that, of the 21438 individual hybridization reaction (3573 validated probes × 6 references strains), 20082 probes (93.67%) yielded the expected result by showing significantly differential signal. Further inspection of each species probe, over 50 % of species probes designed for Lb. acidophilus, Str. thermophilus and Bif. animalis had true-positive signals with target bacteria. Whereas, true-positive signals were lower than 10% for probes designed for Lc. lactis and Leu. mesenteroides. For mixed strain samples, most of significantly positive signals would not be decreased when the amount of target DNA was down to 0.1

並列關鍵字

denaturing gradient gel electrophoresis ； oligonucleotide microarray ； lactic acid bacteria ； identification

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