本文關(guān)鍵詞：金葡菌特異性配基的篩選及初步應(yīng)用　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： SELEX 隨機(jī)ssDNA文庫(kù) 配基 金黃色葡萄球菌 消減篩選

【摘要】： 指數(shù)式富集的配體系統(tǒng)進(jìn)化(SELEX)技術(shù)是近年來發(fā)展起來的研究核酸結(jié)構(gòu)、功能及進(jìn)化的一種新的組合化學(xué)技術(shù)。它在基礎(chǔ)醫(yī)學(xué)和臨床診斷方面具有廣泛的應(yīng)用前景。本研究的目的是建立一種以完整細(xì)菌為靶子的消減SELEX技術(shù),并篩選出與金黃色葡萄球菌特異結(jié)合的一組ssDNA配基(aptamer),為致病微生物的快速檢測(cè)提供理論基礎(chǔ)及實(shí)驗(yàn)依據(jù)。 我們首先設(shè)計(jì)了帶有莖環(huán)的下游引物,通過獲得不同長(zhǎng)度單鏈產(chǎn)物的PCR技術(shù)原理,建立了一種新的ssDNA制備方法。然后采用隨機(jī)區(qū)為45個(gè)堿基、全長(zhǎng)88nt的ssDNA文庫(kù),以金葡菌8325-4菌株為目的靶,鏈球菌A5005菌株和表皮葡萄球菌26069菌株為消減靶,進(jìn)行了七輪的SELEX篩選。對(duì)每一輪文庫(kù)標(biāo)記同位素,結(jié)合實(shí)驗(yàn)證實(shí)第五輪文庫(kù)富集程度最高。將富集的第5輪文庫(kù)進(jìn)行克隆測(cè)序,分別采用MEME在線軟件和RNA STRUCTURE軟件對(duì)序列的結(jié)構(gòu)進(jìn)行分析,得到11條穩(wěn)定序列。隨后利用流式細(xì)胞儀和激光共聚焦顯微鏡找出了5條特異性強(qiáng)的aptamer,其中結(jié)合效果最好的兩個(gè)aptamer的解離常數(shù)均在nM水平。在以上工作的基礎(chǔ)上我們對(duì)組合aptamer的性能進(jìn)行研究,結(jié)果顯示出,與單一的aptamer相比,組合aptamer能夠提高對(duì)金葡菌的敏感性和檢出率。本研究為建立一種新的致病微生物快速檢測(cè)手段奠定了基礎(chǔ)。
[Abstract]:Systematic evolution of ligands by exponential enrichment (SELEX) technology is the study of nucleic acid structure developed in recent years, a new combinatorial chemistry function and evolution. It has wide application prospect in basic medicine and clinical diagnosis. The purpose of this study is to establish a complete SELEX target to reduce bacteria, and selected a group of ssDNA ligand binding with Staphylococcus aureus specific (aptamer), and provide a theoretical basis and experimental basis for the rapid detection of pathogenic microorganisms.
We designed primers with stem loop, the principle of PCR technology to obtain different length single product, establish a new preparation method of ssDNA. Then the random region is 45 bases, ssDNA Library of full-length 88nt, Staphylococcus aureus 8325-4 strain as target, chain coccus aureus strain A5005 and epidermal 26069 strains of Staphylococcus aureus were screened for abatement target, the seven round of the SELEX. For each round of isotope labeling experiments confirmed that the library, combined with the fifth round of the library enriched the highest degree. The fifth round library enrichment were cloned and sequenced, respectively using MEME online software structure and RNA STRUCTURE software for sequence analysis, get 11 stable sequence. Then using flow cytometry and confocal microscopy to identify 5 specificity aptamer, the dissociation constant of binding effect of two aptamer were the best at the level of nM in the above work. Based on our research on the performance of combined aptamer, the results showed that compared with a single aptamer, combined aptamer could improve the sensitivity and detection rate of Staphylococcus aureus. This study laid the foundation for establishing a new rapid detection method for pathogenic microorganisms.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R378

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 余曉峰;嚴(yán)軼;嚴(yán)偉民;姚劍;鄭海松;;SELEX技術(shù)在致病菌檢測(cè)中應(yīng)用進(jìn)展[J];家禽科學(xué);2011年07期

相關(guān)碩士學(xué)位論文 前1條

1 王雷;嗜水氣單胞菌和遲緩愛德華菌適體的SELEX篩選[D];集美大學(xué);2012年

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本文編號(hào)：1387320