本文選題：靶序列富集多重-聚合酶鏈反應(yīng)　切入點(diǎn)：流式熒光雜交檢測(cè)系統(tǒng)　出處：《天津商業(yè)大學(xué)》2015年碩士論文

【摘要】：隨著轉(zhuǎn)基因產(chǎn)業(yè)的迅猛發(fā)展,轉(zhuǎn)基因安全性問(wèn)題逐漸成為公眾關(guān)注的焦點(diǎn)。為此,相關(guān)國(guó)家和地區(qū)采取轉(zhuǎn)基因標(biāo)識(shí)制度,加強(qiáng)對(duì)其監(jiān)管的力度。目前,轉(zhuǎn)基因生物品系繁多、數(shù)量巨大,經(jīng)深加工后其成分又遭到一定程度的破壞,不僅增加了檢測(cè)的工作量,也增加了檢測(cè)的難度。就現(xiàn)有的檢測(cè)手段,早已無(wú)法滿足目前多品系、高通量、高特異、高靈敏的快速檢測(cè)需求。本研究將靶序列富集多重-聚合酶鏈反應(yīng)和流式熒光雜交檢測(cè)系統(tǒng)有機(jī)結(jié)合起來(lái),建立一種在單個(gè)反應(yīng)體系中同時(shí)檢測(cè)五種轉(zhuǎn)基因大豆的方法,實(shí)現(xiàn)包括轉(zhuǎn)基因大豆GTS40-3-2、MON89788、A2704-12、CV127和305423的高通量、高特異和高靈敏的快速檢測(cè)。研究?jī)?nèi)容如下:首先,在Genbank上檢索五種轉(zhuǎn)基因大豆外源基因序列和內(nèi)源基因Lectin序列,通過(guò)i Cubate2.0在線軟件計(jì)算外源基因連接區(qū)域序列的多聚酶親和指數(shù)值(PPI)曲線,并利用Primer Primer5.0、Beacon Designer等軟件設(shè)計(jì)12對(duì)特異性內(nèi)外引物(Fi/Ri和Fo/Ro)、一對(duì)通用超級(jí)引物(Fs/Rs)和六條特異性捕獲探針,其中內(nèi)外引物3′端均位于高PPI值區(qū)域內(nèi),再通過(guò)調(diào)整引物長(zhǎng)度改變Tm值,而內(nèi)引物(Fi/Ri)的5′端分別添加一段能夠被Fs/Rs所識(shí)別的通用序列。其次,建立五種轉(zhuǎn)基因大豆的Tem-PCR反應(yīng)體系。低濃度的Fo/Ro和Fi/Ri只對(duì)靶序列進(jìn)行富集和加標(biāo)簽,高濃度的Fs/Rs對(duì)所有靶序列進(jìn)行指數(shù)擴(kuò)增,克服了常規(guī)多重PCR的高背景、擴(kuò)增效率不一致等難題。最后,建立六種靶序列的流式熒光雜交檢測(cè)體系。Rs的5′端標(biāo)記生物素,其Tem-PCR產(chǎn)物均含有生物素標(biāo)簽,與微球探針進(jìn)行雜交后,可與熒光報(bào)告分子(鏈親和霉素-藻紅素)結(jié)合產(chǎn)生熒光信號(hào)值,經(jīng)Bio-PlexTM 200系統(tǒng)讀取分析,可判斷被檢測(cè)體系中是否含有轉(zhuǎn)基因成分。本研究針對(duì)五種轉(zhuǎn)基因大豆成功建立了基于靶序列富集多重-聚合酶鏈反應(yīng)的流式熒光雜交檢測(cè)系統(tǒng),實(shí)現(xiàn)了五種轉(zhuǎn)基因大豆品系的高通量、高特異、高靈敏(檢測(cè)限可達(dá)0.01%)檢測(cè),重復(fù)性好,五小時(shí)內(nèi)即可完成整個(gè)檢測(cè)過(guò)程,提高了檢測(cè)效率。目前,在國(guó)內(nèi)外將該聯(lián)用技術(shù)用于轉(zhuǎn)基因檢測(cè)鮮有報(bào)道。本研究是將該聯(lián)用技術(shù)用于轉(zhuǎn)基因檢測(cè)的一種探索,初步建立了五種轉(zhuǎn)基因大豆的高通量、高特異、高靈敏的快速檢測(cè)方法,也為其它轉(zhuǎn)基因作物的檢測(cè)研究提供理論借鑒與指導(dǎo)。
[Abstract]:With the rapid development of transgenic industry, the safety of transgenic has gradually become the focus of public attention.To this end, relevant countries and regions to adopt GM marking system, strengthen its regulatory efforts.At present, the number of transgenic organisms is huge, and the components of transgenic organisms are destroyed to a certain extent after deep processing, which not only increases the workload of detection, but also increases the difficulty of detection.The existing detection methods have been unable to meet the current multi-strain, high-throughput, high-specificity, high-sensitive rapid detection requirements.In this study, the target sequence enrichment multiplex polymerase chain reaction (PCR) and flow fluorescence hybridization (FFP) system were combined to establish a method for simultaneous detection of five transgenic soybeans in a single reaction system.High throughput, high specificity and high sensitivity rapid detection including transgenic soybean GTS40-3-2 MON89788A2704-12 CV127 and 305423 were achieved.The main contents are as follows: firstly, five kinds of exogenous gene sequences and endogenous gene Lectin sequences of transgenic soybean were searched on Genbank, and the polymerase affinity index curves were calculated by I Cubate2.0 online software.Using Primer Primer 5.0 and Beacon Designer software, 12 pairs of specific internal and external primer Fir / Ri and For / Roan, a pair of universal super primer Fs / Rs) and six specific capture probes were designed. The 3 'ends of the inner and outer primers were located in the region of high PPI value, and the TM value was changed by adjusting the length of the primers.The 5 'end of the internal primer Fir adds a common sequence that can be recognized by Fs/Rs.Secondly, five Tem-PCR reaction systems of transgenic soybean were established.The low concentration Fo/Ro and Fi/Ri only enrich and label the target sequences, and the high concentration Fs/Rs amplifies all the target sequences exponentially, which overcomes the problems of high background and inconsistent amplification efficiency of conventional multiplex PCR.Finally, the 5 'end labeled biotin of six target sequences was established. The Tem-PCR products of the system contained biotin labels, and were hybridized with microsphere probes.The fluorescent signal value can be generated by combining with fluorescent reporter molecule (chain affinity mycin-phycobiline), and can be read and analyzed by Bio-PlexTM 200 system to determine whether the detected system contains genetically modified components or not.In this study, a flow fluorescence hybridization system based on target sequence enrichment and multiplex polymerase chain reaction (PCR) was successfully established for the detection of five transgenic soybean strains, and the high throughput and specificity of the five transgenic soybean lines were achieved.High sensitivity (detection limit can reach 0.01), good repeatability, the whole detection process can be completed within five hours, improve the detection efficiency.At present, it is seldom reported that the combined technology is used in transgenic detection at home and abroad.This study is an exploration of the combined use of this technology in transgenic detection, and has established five high throughput, high specificity and high sensitivity rapid detection methods for transgenic soybean.It also provides theoretical reference and guidance for the detection of other transgenic crops.
【學(xué)位授予單位】：天津商業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S188

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