應用即時定量聚合&;#37238;連鎖反應於模擬發酵乳中雙歧桿菌之定性與定量檢測

Abstract

許多研究顯示乳酸菌對人體及動物具有保健效果，乳酸菌中又以乳酸桿菌 ( Lactobacillus ) 與雙歧桿菌 ( Bifidobacterium ) 兩屬被廣泛應用於發酵食品、乳製品以及食品或畜牧飼料添加物。早期常以平板計數方法進行菌量計算，但往往會發生低估之可能，即時定量聚合&;#37238;鏈鎖反應 ( real-time quantitative PCR ) 為分子生物技術應用於微生物定量之一大突破，於良好之設計條件下，即時定量 PCR具有更高之靈敏度與準確性。本研究擬應用 PCR 與即時定量 PCR 於檢測發酵乳產品中雙歧桿菌，同時搭配傳統定量方法進行比較，以期建立一有效且可靠之雙歧桿菌定性及定量檢測方法。 本研究以全脂乳混合標準菌株模擬市售之發酵乳產品，樣本DNA 萃取效率將影響即時定量 PCR 之靈敏度與準確性，為萃取完整 DNA，本研究測試三種萃取方法，以核酸偵測 ( A260 /A280 ) 之結果作為判斷依據。定性之部分：菌屬特異性引子組 F_allbif_IS 與 R_allbif_IS 經由 PCR 擴增之目標片段大小為 231 bp；菌種特異性引子組經由 PCR 擴增之目標片段大小介於 67 – 118 bp；定量之部分：以菌種特異性引子組進行即時定量 PCR，得到之 Ct 值對應菌液濃度之對數值繪製成標準曲線，將未知樣品之Ct 值代入標準曲線方程式，即可得知模擬發酵乳中所含之雙歧桿菌菌量。使用 Student’s t-test 比較以即時定量 PCR 得到之菌量與培養基培養之結果 ( p > 0.05 )，兩法間並無顯著差異。傳統培養定量之時間需求為2 - 7天，而以分子生物技術操作時間則縮短至八小時內完成，利用 culture-independent 之菌量計數具有應用之潛力。 本研究成功建立一快速鑑定與定量發酵乳中雙歧桿菌之方法，以PCR反應可區分發酵乳中雙歧桿菌菌種，以即時定量 PCR 可檢測發酵乳中雙歧桿菌菌量，更可提供未來研究之發展基礎。

Lactic acid bacteria ( LAB ) strains with probiotics functions have been used for the processing of fermented food, milk products as well as food and feed supplements. Initial people used plate counts method to enumerate bacteria, but it often causes underestimation. Real-time quantitative PCR is a newly molecular technique that can be used to enumerate bacteria. Many studies indicated that real-time quantitative PCR with good operating conditions provided high sensitivity and accuracy. For quantitative analysis, we used PCR and real-time quantitative PCR to indentified and quantitative bifidobacteria in simulated fermented milk. At the same time, we also used plate count method to compare with real-time quantitative PCR. This study highlighted the advantage of real-time quantitative PCR and investigated the detection of simulated fermented milk products contain bifidobacteria. The simulated fermented milk are composed of whole milk and bifidobacteria reference strains. For extracting complete DNA, we tried three methods and determine to the value of A260 /A280. For Qualitative analysis , PCR products were 231 bp by genus-specific primers F_allbif_IS and R_allbif_IS ; PCR products were 67 to 118 bp by species-specific primers, respectively. For quantitative, real-time quantitative PCR performed with species-specific primers to analyze 7 reference strains in simulated fermented milk products. By using the Ct (cycle threshold) and the concentration of bacteria cells could generate the standard curve of bifidobacteria reference strains. Substitution Ct value of simulated fermented milk into standard curve could evaluate the concentration of bacteria. The Student's t-test was used to compare each quantitative analysis between plating enumeration and real-time quantitative PCR. The result (p > 0.05) indicated that there was no significant difference between the two methods at a confidence level of 95 %. Besides, plate count method spent more time than real-time quantitative PCR. This study describes a detection method for bifidobacteria in simulated fermented milk. The PCR analysis combined species -specific PCR is showing a great detection and identification potential using for seven species of bifidobacteria. The species-specific real-time quantitative PCR for evaluating the concentration of bacteria is no significant difference with the plate count method for enumeration. The result indicated that it has potential for developing a culture-independent bacteria enumeration procedure and set a foundation for future studies.