本文關(guān)鍵詞： G-四鏈體 級(jí)聯(lián)信號(hào)擴(kuò)增 可視化 轉(zhuǎn)基因檢測(cè)　出處：《合肥工業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：伴隨著生物技術(shù)的進(jìn)步,轉(zhuǎn)基因作物在全球范圍內(nèi)得到廣泛的種植。轉(zhuǎn)基因技術(shù)打破了生殖隔離的限制,使得轉(zhuǎn)基因作物整合了多物種的基因。雖然未有科學(xué)數(shù)據(jù)直接證明,但是轉(zhuǎn)入的外源基因仍然對(duì)環(huán)境和食品安全構(gòu)成潛在的威脅。所以對(duì)于轉(zhuǎn)基因作物以及相關(guān)產(chǎn)品的安全性評(píng)價(jià)、監(jiān)管和檢測(cè)顯得尤為重要。本論文主要研究利用新型核苷酸探針和DNA擴(kuò)增方法聯(lián)用對(duì)轉(zhuǎn)基因成分進(jìn)行檢測(cè)的方法。在研究過程中,使用核苷酸探針與DNA擴(kuò)增方法聯(lián)合使用,并探索出合適的體系,以及聯(lián)用體系在不同條件下的通用性。通過設(shè)計(jì)一段包含G-四鏈體互補(bǔ)序列的特異性探針用于等溫?cái)U(kuò)增方法,對(duì)含Bt基因部分序列的單鏈DNA分子進(jìn)行可視化檢測(cè)。通過優(yōu)化,確定了1μL 10X Isothermal Amplification和0.5μL 10X NEBuffer 3.1、10mM Mg~(2+)、4μM Hemin、5U Nb.BbvCI、4U Bst 2.0 WarmStar,并使用三片層的分子內(nèi)G-四鏈體探針作為最優(yōu)反應(yīng)體系,孵育60min完成對(duì)目的基因的檢測(cè)。建立了一種簡(jiǎn)單的,無需標(biāo)記的G-四鏈體等溫?cái)U(kuò)增檢測(cè)體系。進(jìn)一步通過將G-四鏈體核苷酸探針和兩種DNA擴(kuò)增方法聯(lián)用。以轉(zhuǎn)基因水稻科豐6號(hào)基因組DNA作為對(duì)cry1Ab/cry1Ac基因檢測(cè)的目標(biāo)DNA,為了降低背景信號(hào)對(duì)檢測(cè)結(jié)果的影響,使用初始1μM濃度的含有分子內(nèi)G-四鏈體結(jié)構(gòu)的引物進(jìn)行PCR反應(yīng)。當(dāng)體系內(nèi)存在存在cry1Ab/cry1Ac基因的植物基因組DNA時(shí),會(huì)通過PCR和等溫?cái)U(kuò)增的反應(yīng)過程,使體系內(nèi)積累大量的G-四鏈體,并通過顯色系統(tǒng)使本來無色的溶液變成藍(lán)綠色。基于此,建立了一種可以直接從轉(zhuǎn)基因?qū)嶋H樣品中檢測(cè)cry1Ab/cry1Ac基因的可視化檢測(cè)方法。除此之外,還探索了級(jí)聯(lián)擴(kuò)增方法在檢測(cè)其他轉(zhuǎn)基因作物樣品時(shí),特別是具有復(fù)雜基因組結(jié)構(gòu)的轉(zhuǎn)基因玉米MON89034的檢測(cè)效果。使用5μL和2μL的對(duì)應(yīng)各自檢測(cè)體統(tǒng)的引物分別用于Nb.BbvCI和Nt.BstNBI檢測(cè)體系的PCR過程,之后再分別在37℃和55℃酶的最適溫度下孵育30min。最終Nb.BbvCI體系和Nt.BstNBI體系分別對(duì)MON89034的檢出限為100拷貝和50拷貝,達(dá)到了轉(zhuǎn)基因檢測(cè)的要求。因此,建立了一種選擇性好,并且具有通用性的可視化轉(zhuǎn)基因檢測(cè)方法。
[Abstract]:With advances in biotechnology, genetically modified crops have been widely planted worldwide. Transgenic technologies have broken the restrictions of reproductive isolation and led to the integration of multi-species genes into genetically modified crops, although there is no direct scientific data to prove it. But the foreign genes still pose a potential threat to the environment and food safety. In this paper, we mainly study the method of detecting transgenic components by using new nucleotide probe and DNA amplification method. In the process of research, we use nucleotide probe and DNA amplification method. The suitable system and the versatility of the combined system under different conditions were explored. A specific probe containing the complementary sequence of G-quadruplex was designed for isothermal amplification. Visual detection of single-stranded DNA molecule containing partial sequence of BT gene was carried out. Through optimization, 1 渭 L 10X Isothermal Amplification and 0.5 渭 L 10X NEBuffer 3.1mM Mg~(2 were determined as 4 渭 M Hemin5U NB. BbvCI4U Bst 2.0 WarmStar.Three layers of intramolecular G-quad probe were used as the optimal reaction system. After incubation for 60 minutes, the target gene was detected. The G-quad nucleotide probe and two DNA amplification methods were further used to detect the cry1Ab/cry1Ac gene in transgenic rice Kefeng 6 genomic DNA. In order to reduce the influence of background signal on the detection results, The PCR reaction was carried out by using primer containing intramolecular G- quadruplex structure with initial concentration of 1 渭 M. When there was plant genomic DNA with cry1Ab/cry1Ac gene in the system, it would accumulate a large number of G- quartiles through the reaction process of PCR and isothermal amplification. Based on this, a visualized method for detecting cry1Ab/cry1Ac gene directly from actual transgenic samples was established. The cascaded amplification method was also explored in the detection of other transgenic crop samples, In particular, the detection effect of transgenic maize MON89034 with complex genomic structure was studied. 5 渭 L and 2 渭 L primers were used to detect the PCR process of Nb.BbvCI and Nt.BstNBI detection system, respectively. Then incubated at the optimum temperature of 37 鈩，

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