本文關鍵詞： G-四鏈體 級聯(lián)信號擴增 可視化 轉基因檢測　出處：《合肥工業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：伴隨著生物技術的進步,轉基因作物在全球范圍內(nèi)得到廣泛的種植。轉基因技術打破了生殖隔離的限制,使得轉基因作物整合了多物種的基因。雖然未有科學數(shù)據(jù)直接證明,但是轉入的外源基因仍然對環(huán)境和食品安全構成潛在的威脅。所以對于轉基因作物以及相關產(chǎn)品的安全性評價、監(jiān)管和檢測顯得尤為重要。本論文主要研究利用新型核苷酸探針和DNA擴增方法聯(lián)用對轉基因成分進行檢測的方法。在研究過程中,使用核苷酸探針與DNA擴增方法聯(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。通過設計一段包含G-四鏈體互補序列的特異性探針用于等溫擴增方法,對含Bt基因部分序列的單鏈DNA分子進行可視化檢測。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)反應體系,孵育60min完成對目的基因的檢測。建立了一種簡單的,無需標記的G-四鏈體等溫擴增檢測體系。進一步通過將G-四鏈體核苷酸探針和兩種DNA擴增方法聯(lián)用。以轉基因水稻科豐6號基因組DNA作為對cry1Ab/cry1Ac基因檢測的目標DNA,為了降低背景信號對檢測結果的影響,使用初始1μM濃度的含有分子內(nèi)G-四鏈體結構的引物進行PCR反應。當體系內(nèi)存在存在cry1Ab/cry1Ac基因的植物基因組DNA時,會通過PCR和等溫擴增的反應過程,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綠色�；诖�,建立了一種可以直接從轉基因?qū)嶋H樣品中檢測cry1Ab/cry1Ac基因的可視化檢測方法。除此之外,還探索了級聯(lián)擴增方法在檢測其他轉基因作物樣品時,特別是具有復雜基因組結構的轉基因玉米MON89034的檢測效果。使用5μL和2μL的對應各自檢測體統(tǒng)的引物分別用于Nb.BbvCI和Nt.BstNBI檢測體系的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MON89034的檢出限為100拷貝和50拷貝,達到了轉基因檢測的要求。因此,建立了一種選擇性好,并且具有通用性的可視化轉基因檢測方法。
[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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