本文關(guān)鍵詞： 指數(shù)富集配體的系統(tǒng)進(jìn)化技術(shù) 寡核苷酸適配子 化膿鏈球菌 無(wú)乳鏈球菌 磁性納米材料　出處：《江南大學(xué)》2013年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：指數(shù)富集配體的系統(tǒng)進(jìn)化技術(shù)（Systematic Evolution of Ligands by ExponentialEnrichment，SELEX），是90年代初研制成功的一種新的組合化學(xué)技術(shù)。適配子（Aptamer）實(shí)質(zhì)是利用SELEX技術(shù)，從體外人工合成的隨機(jī)寡核苷酸（ssDNA）文庫(kù)中經(jīng)過(guò)數(shù)輪反復(fù)篩選，得到的與靶分子具有極高特異性和親和力的一段寡核苷酸序列。目前，已報(bào)道的食源性致病菌的檢測(cè)方法多種多樣，但各有其優(yōu)缺點(diǎn)。本研究通過(guò)細(xì)胞SELEX技術(shù)（Cell-SELEX）篩選，得到了兩種致病菌（化膿鏈球菌和無(wú)乳鏈球菌）的寡核苷酸適配子，并且建立了基于寡核苷酸適配子識(shí)別的新型檢測(cè)技術(shù)。 本文首先體外合成一個(gè)80nt，中間40個(gè)隨機(jī)堿基序列的ssDNA文庫(kù)，以化膿鏈球菌為靶標(biāo)，通過(guò)Cell-SELEX技術(shù)及反篩SELEX技術(shù)（counter SELEX）進(jìn)行12輪重復(fù)篩選，將篩選得到的DNA序列進(jìn)行克隆、測(cè)序。運(yùn)用DNAMAN軟件、RNAstructure軟件對(duì)其進(jìn)行一級(jí)結(jié)構(gòu)及二級(jí)結(jié)構(gòu)分析；通過(guò)羧基熒光素（FAM）標(biāo)記篩選得到的ssDNA適配子，運(yùn)用流式細(xì)胞術(shù)考察了適配子與目標(biāo)化膿鏈球菌適配子結(jié)合的親和力和特異性，篩選得到兩條親和力高、特異性強(qiáng)的適配子，其解離常數(shù)分別為44.25±5.203nmol/L、53.89±7.586nmol/L，與化膿鏈球菌的結(jié)合率為77.05%、72.65%。 同時(shí)，體外合成另一個(gè)相同長(zhǎng)度、不同引物的寡核苷酸文庫(kù)，，以無(wú)乳鏈球菌為靶標(biāo)，采用與化膿鏈球菌類(lèi)似的方法得到了可以與無(wú)乳鏈球菌特異結(jié)合的7條寡核苷酸適配子，對(duì)其進(jìn)行FAM熒光標(biāo)記，通過(guò)流式細(xì)胞術(shù)從中篩選得到兩條高親和力、高特異性適配子，與無(wú)乳鏈球菌的結(jié)合率分別為77.33%、80.95%，解離常數(shù)為78.27±12.85nmol/L，49.88±6.871nmol/L。 隨后，本研究以磁珠-適配子復(fù)合物為捕獲探針，以FAM熒光素標(biāo)記的適配子為顯示探針，成功構(gòu)建了靈敏、快捷的食源性致病菌熒光檢測(cè)新方法。在實(shí)驗(yàn)優(yōu)化條件下，化膿鏈球菌菌落個(gè)數(shù)在70-7100cfu/mL范圍內(nèi)時(shí)，與熒光強(qiáng)度呈良好線性關(guān)系，檢出限為70cfu/mL，牛奶樣品中的加標(biāo)回收率為83.9%-103.3%；無(wú)乳鏈球菌菌落個(gè)數(shù)在50-5200cfu/mL范圍內(nèi)時(shí)，與熒光強(qiáng)度呈良好線性關(guān)系，檢出限為50cfu/mL，牛奶樣品中的加標(biāo)回收率為95.2%-102.5%。
[Abstract]:Systematic Evolution of Ligands by ExponentialEnrichmentSelex, a new combinatorial chemical technique developed at the beginning of 90s, is based on the use of SELEX technique. A sequence of oligonucleotides with high specificity and affinity to the target molecule was obtained from the in vitro synthetic random oligonucleotide ssDNA library after several rounds of repeated screening. The reported methods for the detection of foodborne pathogenic bacteria are various, but each has its own advantages and disadvantages. In this study, the oligonucleotide aptamers of two pathogenic bacteria (Streptococcus pyogenes and Streptococcus lactobacillus) were obtained by cell SELEX technique (Cell-SELEX). A new detection technique based on oligonucleotide aptamer recognition was established. In this paper, we first synthesized an 80nt ssDNA library with 40 random base sequences in vitro. Using Streptococcus pyogenes as the target, we carried out 12 rounds of repeated screening by Cell-SELEX and reverse sieve SELEX technique, and cloned the selected DNA sequence. Sequencing. The primary structure and secondary structure of DNAMAN were analyzed by DNAMAN software. The ssDNA adaptor was screened by carboxyl fluorescein (FAM) labeling. The affinity and specificity of aptamer combined with target Streptococcus pyogenes aptamer were investigated by flow cytometry. Two aptamers with high affinity and strong specificity were obtained. Their dissociation constants were 44.25 鹵5.203 nmol / L (53.89 鹵7.586nmol / L), and the binding rate with Streptococcus pyogenes was 77.05nmol / L (77.05nmol / L). At the same time, another oligonucleotide library of the same length and different primers was synthesized in vitro. Using Streptococcus lactis as the target, seven oligonucleotide adapters were obtained by using a method similar to that of Streptococcus pyogenes. Two high affinity, high specific aptamers were screened by flow cytometry, and the binding rate of them to Streptococcus lactis was 77.33nmol / L. The dissociation constant was 78.27 鹵12.85nmol / L (49.88 鹵6.871 nmol / L), and the binding rates were 77.33nmol / L and 80.95nmol / L, respectively, and the dissociation constant was 78.27 鹵12.85nmol / L ~ + 49.88 鹵6.871 nmol / L respectively. Subsequently, a sensitive and fast fluorescent detection method for foodborne pathogenic bacteria was successfully constructed using magnetic bead aptamer complex as capture probe and aptamer labeled with FAM fluorescein as display probe. When the colony number of Streptococcus pyogenes was within the range of 70-7100 cfur / mL, it had a good linear relationship with the fluorescence intensity, the detection limit was 70 cfumL, the recovery rate in milk samples was 83.9 -103.3%, and the number of Streptococcus pyogenes colonies in the range of 50-5200 cfur / mL showed a good linear relationship with the fluorescence intensity. The detection limit was 50cfu-mL, and the recoveries in milk samples were 95.2- 102.5.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R155.5

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