Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/25570
標題: 應用變性梯度度膠體電泳探討肉雞消化道乳酸桿菌相之變化
Monitoring the Lactobacillus population in broiler gastrointestinal tract by Denaturing Gradient Gel Electrophoresis
作者: 張文欣
Chang, Wen-Hsin
關鍵字: 肉雞;broiler;變性梯度膠體電泳;菌相;Polymerase Chain Reaction- Denaturing Gradient Gel Electrophoresis;microflora
出版社: 動物科學系所
摘要: 
動物腸道菌相為健康的指標,其易受到宿主年齡及飼糧組成等因子所影響,而菌相組成、菌數的改變及菌種的分類在微生物學的研究已有許多方法可以量化及質化。在環境微生物相的研究上常採用PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis)電泳技術,以一種較簡便及巨觀的方式探討環境中主要菌群的組成及改變。因此,本研究目的為(1)建立腸道中乳酸桿菌相變化之PCR-DGGE電泳條件,(2)使用此技術監測年齡改變及益生菌對肉雞消化道中乳酸桿菌相之影響。結果顯示,雞隻腸道內容物樣品選用乳酸桿菌專一性引子對(LabtufGC及LabtufR),經巢式PCR (nested PCR)擴增並純化後,於含10%丙烯醯胺且變性梯度為35-47.5%之膠體,在60℃下以80V進行16小時之電泳後經銀染方式進行染色,可獲得條帶分離效果較佳及解析度較高之電泳圖譜。而初生雛口服三天乳酸桿菌液後犧牲並蒐集消化道內容物進行PCR-DGGE,在嗉囊及迴腸內容物DGGE圖譜上均可檢測到對照組未出現之乳酸桿菌條帶,顯示PCR-DGGE技術於探討家禽消化道乳酸桿菌相之變化有一定的可行性。進一步於雞隻飼糧中添加乳酸桿菌粉,於不同日齡犧牲後蒐集消化道內容物進行PCR-DGGE電泳,結果顯示,雞隻隨著年齡增長,迴腸內容物DGGE圖譜上條帶數目隨著日齡增加逐漸減少,乳酸桿菌種歧異度(Shannon’s index)亦隨之降低,於1日齡時為3.262,至35日齡時降低為2.237,但在盲腸反之,隨年齡增長則乳酸桿菌相歧異度增加,由1日齡之2.753至35日齡時提高為3.738。另外,由相似度指數結果可得知,不同消化道部位乳酸桿菌的發育程度均有所不同,嗉囊在1日齡以後菌相趨於穩定,而迴腸菌相則在7日齡後較無改變,至於盲腸部分菌相隨年齡增長改變,至採樣後期(35日齡)仍持續變化。另外,添加益生菌不會影響嗉囊中乳酸桿菌的種數,但可能提高菌種間的均勻度。益生菌可增加21日齡時盲腸乳酸桿菌條帶數,進而提升菌相歧異度之效果。益生菌和抗生素對促進宿主生長性狀之機制不完全相同,抗生素主要藉由減少腸道中互相競爭之細菌種類,降低腸道菌相之歧異度而達到減少能量浪費之作用,而給予益生菌處理可提高嗉囊及迴腸乳酸桿菌相之歧異度,藉由有益菌的均勻度增加,使消化道菌相趨於正相平衡,進一步達到促進生長之效果。綜上所述,PCR-DGGE技術可應用於動物腸道乳酸桿菌相之監測,未來可配合分子選殖技術進一步判定圖譜條帶所代表之菌種,並藉由不同引子對的組合,更清楚的了解腸道中複雜菌相的改變。

An understanding of the dynamics of bacterial community structure in the chicken intestine is essential for selection of diets for optimal nutrition, effective treatment of enteric pathogens, and the development of competitive exclusion products. The microbial diversity, count of bacteria and classification of bacteria in microbiology are many ways to quantitative and qualitative. It's used in research on PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis), that a more convenient way to explore the changes in the composition of flora. In the present study, establishing the optimal condition of PCR-DGGE, and investigate the transition of the bacterial community structure and the predominant bacteria in the intestine of chicks from hatching to 5 wk of age or diet supplement antibiotic and probiotic were investigated using PCR-DGGE and phylogenetic analysis. The results demonstrated that samples of intestinal contents used lactobacillus-specific primers (LabtufGC / LabtufR), by nested PCR amplification and purification, with 10% acrylamide and denaturing gradient of 35-47.5% of the gel, in order to 80V at 60 ℃ for 16 hours after electrophoresis were stained by the silver staining method, obtain a better separation bands and higher resolution of DGGE profile. Newborn chicks after oral lactobacillus broth three days, digestive tract contents collected for PCR-DGGE, the crop and ileal contents in the DGGE patterns can be detected the bands of lactic acid bacteria that not appear in the control group. With age, the bands number of ileal contents DGGE profiles were decreased, and the biodiversity of Lactobacillus species (Shannon''s index) also decreases. However, the opposite in the cecum, the biodiversity of Lactobacillus species were increase with age. Index of similarity results that the development of lactic acid bacteria were different on disparity parts of the gastrointestinal tract. Crop microflora stabilized after 1-day-old, and ileum microflora in no change after 7 days, as cecal microflora changes due to age, to the late sampling (35 days) continued to vary. Probiotics and antibiotics to promote growth performent of the host mechanisms are not identical, that antibiotics to decrease intestinal bacterial species and reducing the biodiversity of intestinal microflora and to reduce energy waste, the probiotic can improve the biodiversity of lactobacillus on crop and ileum, and the digestive tract microflora to become positive equilibrium, and further to improve the growth performent. Application of PCR-DGGE to monitor the changes of Lactobacillus population in gastrointestinal tract of broiler is feasible. The species of Lactobacillus can be determined by PCR-DGGE combine with molecular cloning technique in the further study.
URI: http://hdl.handle.net/11455/25570
Appears in Collections:動物科學系

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