转基因大豆流式荧光杂交检测体系的研究

发布时间：2018-04-05 00:22

本文选题：靶序列富集多重-聚合酶链反应　切入点：流式荧光杂交检测系统　出处：《天津商业大学》2015年硕士论文

【摘要】：随着转基因产业的迅猛发展,转基因安全性问题逐渐成为公众关注的焦点。为此,相关国家和地区采取转基因标识制度,加强对其监管的力度。目前,转基因生物品系繁多、数量巨大,经深加工后其成分又遭到一定程度的破坏,不仅增加了检测的工作量,也增加了检测的难度。就现有的检测手段,早已无法满足目前多品系、高通量、高特异、高灵敏的快速检测需求。本研究将靶序列富集多重-聚合酶链反应和流式荧光杂交检测系统有机结合起来,建立一种在单个反应体系中同时检测五种转基因大豆的方法,实现包括转基因大豆GTS40-3-2、MON89788、A2704-12、CV127和305423的高通量、高特异和高灵敏的快速检测。研究内容如下:首先,在Genbank上检索五种转基因大豆外源基因序列和内源基因Lectin序列,通过i Cubate2.0在线软件计算外源基因连接区域序列的多聚酶亲和指数值(PPI)曲线,并利用Primer Primer5.0、Beacon Designer等软件设计12对特异性内外引物(Fi/Ri和Fo/Ro)、一对通用超级引物(Fs/Rs)和六条特异性捕获探针,其中内外引物3′端均位于高PPI值区域内,再通过调整引物长度改变Tm值,而内引物(Fi/Ri)的5′端分别添加一段能够被Fs/Rs所识别的通用序列。其次,建立五种转基因大豆的Tem-PCR反应体系。低浓度的Fo/Ro和Fi/Ri只对靶序列进行富集和加标签,高浓度的Fs/Rs对所有靶序列进行指数扩增,克服了常规多重PCR的高背景、扩增效率不一致等难题。最后,建立六种靶序列的流式荧光杂交检测体系。Rs的5′端标记生物素,其Tem-PCR产物均含有生物素标签,与微球探针进行杂交后,可与荧光报告分子(链亲和霉素-藻红素)结合产生荧光信号值,经Bio-PlexTM 200系统读取分析,可判断被检测体系中是否含有转基因成分。本研究针对五种转基因大豆成功建立了基于靶序列富集多重-聚合酶链反应的流式荧光杂交检测系统,实现了五种转基因大豆品系的高通量、高特异、高灵敏(检测限可达0.01%)检测,重复性好,五小时内即可完成整个检测过程,提高了检测效率。目前,在国内外将该联用技术用于转基因检测鲜有报道。本研究是将该联用技术用于转基因检测的一种探索,初步建立了五种转基因大豆的高通量、高特异、高灵敏的快速检测方法,也为其它转基因作物的检测研究提供理论借鉴与指导。
[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.
【学位授予单位】：天津商业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S188

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