本文關(guān)鍵詞：鼠傷寒沙門氏菌適配體的篩選及應(yīng)用研究　出處：《湖南師范大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鼠傷寒沙門氏菌 適配體篩選 氧化石墨烯 微生物檢測(cè)

【摘要】：沙門氏菌病是公共衛(wèi)生學(xué)上最有重要意義的的人畜共患病之一,其病原沙門氏菌屬于腸桿菌科,是一大群寄生于人類和動(dòng)物腸道內(nèi),抗原構(gòu)造和生化反應(yīng)相類似的革蘭氏陰性桿菌的統(tǒng)稱。尤其以鼠傷寒沙門氏菌為代表的病原菌在食品安全、環(huán)境監(jiān)測(cè)、疾病預(yù)防等方面造成了巨大的威脅,對(duì)人體健康造成了很大危害。如何實(shí)現(xiàn)沙門氏菌的快速檢測(cè)成為當(dāng)前研究的熱點(diǎn)問題。 適配體是通過指數(shù)富集的配體系統(tǒng)進(jìn)化技術(shù)(SELEX)篩選得到的一段短的單鏈DNA或RNA序列,它能夠與相應(yīng)的靶標(biāo)分子如金屬離子、有機(jī)小分子藥物、蛋白質(zhì)乃至整個(gè)細(xì)胞進(jìn)行高親和力和強(qiáng)特異性的結(jié)合。由于適配體幾乎可與每一個(gè)目標(biāo)分子進(jìn)行化學(xué)修飾,在生物分子的分析檢測(cè)和治療診斷等方面開創(chuàng)了一個(gè)新的局面。 石墨烯是一種由純碳原子緊密堆積成單層蜂窩網(wǎng)狀結(jié)構(gòu)的新型二維納米材料。由于具有非常大的比表面積、優(yōu)良的導(dǎo)熱性和導(dǎo)電性、使得石墨烯在諸多領(lǐng)域都得到了廣泛應(yīng)用。氧化石墨烯是石墨烯的一種重要衍生物,表面大量存在的含氧基團(tuán)能夠增加其親和性及水溶性。近年來,通過利用氧化石墨烯良好的生物相容性、高的熒光猝滅效率以及大的比表面積等優(yōu)點(diǎn),相繼開發(fā)了各種熒光生物傳感器。 本論文以鼠傷寒沙門氏菌特異性識(shí)別、靈敏檢測(cè)這一熱點(diǎn)問題為切入點(diǎn),主要開展了以下兩個(gè)方面的研究工作： 一、SELEX篩選鼠傷寒沙門氏菌適配體方法的建立及適配體克隆、測(cè)序、鑒定及分析 以鼠傷寒沙門氏菌(S. typhimurium)為靶標(biāo),利用體外細(xì)胞SELEX篩選技術(shù),構(gòu)建了全長(zhǎng)為78個(gè)堿基,中間含35個(gè)堿基隨機(jī)序列的單鏈核苷酸文庫(kù),通過11輪反復(fù)常規(guī)篩選以及以腸炎、豬霍亂、甲型副傷寒和亞利桑那沙門氏菌為靶標(biāo)的4輪消減篩選,初步篩選到一組能特異性結(jié)合S. typhimurium的核酸適配體群。對(duì)最后一輪篩選得到的核酸適配體的PCR產(chǎn)物進(jìn)行克隆測(cè)序,獲得了39條適配體序列。隨后對(duì)它們進(jìn)行一級(jí)結(jié)構(gòu)的同源性分析及二級(jí)結(jié)構(gòu)的模擬分析,最終獲得了6條對(duì)S. typhimurium親和力高、特異性好的序列。利用熒光分析法對(duì)其親和力進(jìn)行測(cè)定,Kd值均為0～100nM,其中Apt6的親和力最高,Kd值僅為30±4nM。隨后通過選擇特異性實(shí)驗(yàn)分析表明,適配體與S. typhimurium的結(jié)合具有很強(qiáng)的特異性且對(duì)同種屬的沙門氏菌不會(huì)有交叉結(jié)合。 二、構(gòu)建基于納米材料氧化石墨烯的熒光適配體傳感器 利用氧化石墨烯熒光猝滅劑的性質(zhì)和高特異性適配體,構(gòu)建了快速、簡(jiǎn)單、高靈敏的熒光適配體傳感器。在沒有靶標(biāo)S. typhimurium時(shí),熒光標(biāo)記的適配體會(huì)自動(dòng)吸附到氧化石墨烯表面,適配體與氧化石墨烯之間發(fā)生熒光能量共振轉(zhuǎn)移(FRET),適配體的熒光由于能量轉(zhuǎn)移被迅速猝滅；當(dāng)加入靶標(biāo)菌后,適配體會(huì)與靶標(biāo)形成特殊的空間結(jié)構(gòu),從而使適配體從氧化石墨烯表面釋放出來,熒光恢復(fù)。結(jié)果表明這種傳感器的檢測(cè)下限為102cfu/mL,并在103—108cfu/mL濃度之間呈線性關(guān)系,同時(shí)有很高的選擇性。因此,這種基于氧化石墨烯的熒光適配體傳感器可以有效的用于病原菌的快速檢測(cè)。
[Abstract]:Salmonellosis is one of the most important public health is the zoonotic disease of the pathogenic Salmonella bacteria belonging to the Enterobacteriaceae, is a large group of parasites in the intestinal tract of human and animal, referred to as the antigenic structure and biochemical reactions similar to gram negative bacteria. Especially the pathogens in Salmonella typhimurium as a representative in food safety, environmental monitoring, disease prevention and other aspects caused a huge threat, causing great harm to human health. How to achieve the rapid detection of Salmonella has become a hot issue in current research.
Aptamers are through the evolution of ligands by exponential enrichment system (SELEX) screened a short single stranded DNA or RNA sequence, it can with the corresponding target molecules such as metal ions, small organic molecules, and the whole cell binding protein with high affinity and high specificity. The aptamer almost by chemical modification and each target molecule in biomolecular detection and analysis of diagnosis and treatment so as to create a new situation.
Graphene is a pure carbon atoms tightly packed into a two-dimensional model of nano materials. Due to single-layer cellular network structure has a very large surface area, thermal conductivity and excellent conductivity of graphene, which has been widely applied in many fields. The graphene oxide is an important derivative of Shi Moxi, oxygen there are a large number of surface groups can increase their affinity and solubility in water. In recent years, through the use of graphene oxide with good biocompatibility, fluorescence quenching of high efficiency and large surface area and so on, have developed a variety of fluorescence biosensor.
In this paper, the specific identification of Salmonella typhimurium, sensitive detection of this hot issue as the breakthrough point, mainly carried out the following two aspects of the research work:
Establishment of a SELEX screening method for Salmonella typhimurium and its aptamer cloning, sequencing, identification and analysis
Salmonella typhimurium (S. typhimurium) as the target, using in vitro SELEX screening technology, constructed a full-length 78 BP single nucleotide intermediate library containing 35 BP random sequence, through 11 rounds of repeated routine screening as well as enteritis, hog cholera, Salmonella paratyphi A and Alexander Sangnashamenshi bacteria as the targets of the 4 round subtractive screening, preliminary screening to a set of specific binding S. typhimurium nucleic acid aptamer group. PCR products of the last round of nucleic acid aptamers were cloned and sequenced, obtained 39 aptamer sequences. Then simulate the homology analysis of primary structure and secondary structure of two of them. Won the 6 S. typhimurium of high affinity and specificity. The sequence of their affinity was determined by fluorescence analysis method, Kd value was 0 ~ 100nM, which Apt6 the highest affinity, Kd value is only 30 4N. M. followed by specific experimental analysis showed that the combination of aptamers and S. typhimurium was highly specific and had no cross linking to Salmonella belonging to the same genus.
Two, a fluorescent aptamer sensor based on nanomaterial graphene oxide
By using properties of graphene oxide fluorescence quenching and high specificity of aptamer constructs, rapid, simple, high sensitive fluorescent aptamer sensor. In the absence of target S. typhimurium, fluorescence labeled adaptation of automatic adsorption onto graphene oxide and graphene oxide between the aptamer and fluorescence resonance energy transfer (FRET), aptamer fluorescence energy transfer due to rapid quenching; when added to the target bacteria after adaptation of formation of special structures and targets, so that the aptamer released from the surface of graphene oxide, fluorescence recovery. The results showed that the detection limit of the sensor is 102cfu/mL, and a linear relationship between the 103 108cfu/mL concentration, and has very high selectivity. Therefore, the fluorescence of graphene oxide based on aptamer sensor can be used for rapid detection of pathogenic bacteria effectively.

【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張艷紅,吳延功,杜元釗,黃素珍;沙門氏菌快速檢測(cè)方法研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2001年02期

2 王巍;賈凌云;;適配體篩選方法研究進(jìn)展[J];分析化學(xué);2009年03期

3 段諾;吳世嘉;王周平;;核酸適配體識(shí)別-熒光法檢測(cè)赭曲霉素A[J];分析化學(xué);2011年03期

4 王聰艷;孫偉;李建國(guó);裴玉賀;蔡民華;晏月明;;SELEX技術(shù)應(yīng)用研究進(jìn)展[J];生物技術(shù)通報(bào);2006年S1期

5 謝小燕,夏寧邵;生物學(xué)中熒光共振能量轉(zhuǎn)移的研究應(yīng)用進(jìn)展[J];生物技術(shù)通訊;2001年03期

6 李衛(wèi)濱;蘭小鵬;楊湘越;吳文冰;;用SELEX技術(shù)篩選環(huán)孢霉素A適體[J];生物技術(shù)通訊;2007年05期

7 S. KUMAR;K. BALAKRISHNA;HV. BATRA;;Enrichment-ELISA for Detection of Salmonella typhi From Food and Water Samples[J];Biomedical and Environmental Sciences;2008年02期

8 王高產(chǎn);孫振鈞;;沙門氏菌的檢測(cè)方法[J];豬業(yè)科學(xué);2009年12期

9 范宏英,吳清平,吳若菁,寇曉霞,張菊梅,吳慧清;飲用水中5種致病菌多重PCR技術(shù)檢測(cè)研究[J];微生物學(xué)通報(bào);2005年03期

10 陶軍,張樹宏,吳仲梁;“自動(dòng)熒光酶標(biāo)免疫測(cè)試儀”與常規(guī)培養(yǎng)法對(duì)凍禽肉中沙門氏菌的檢測(cè)效果的比較[J];現(xiàn)代科學(xué)儀器;2001年03期

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