本文選題：多重PCR　切入點(diǎn)：轉(zhuǎn)基因水稻　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：隨著全球轉(zhuǎn)基因作物及其產(chǎn)品的數(shù)量和種類越來越多,其所帶來的潛在風(fēng)險(xiǎn)也備受人們的關(guān)注。轉(zhuǎn)基因生物安全監(jiān)管事關(guān)糧食安全、食品安全和生態(tài)安全,以歐盟為代表的國家和地區(qū)出臺了一系列的法律法規(guī),加強(qiáng)監(jiān)管。轉(zhuǎn)基因成分檢測是轉(zhuǎn)基因生物安全監(jiān)管的重要技術(shù)支撐和手段。近年來,基于傳統(tǒng)PCR檢測技術(shù)已經(jīng)不能滿足轉(zhuǎn)基因成分檢測簡便快捷高效日益發(fā)展的需求。多重PCR(Multiplex PCR)技術(shù)因特異性強(qiáng)、靈敏度高、成本低、通量高、節(jié)省實(shí)驗(yàn)樣品和藥品等諸多優(yōu)點(diǎn),已經(jīng)在轉(zhuǎn)基因成分檢測研究領(lǐng)域得到了廣泛的研究和應(yīng)用。本文以轉(zhuǎn)基因水稻TT51-1、KMD1、KF6和轉(zhuǎn)基因油菜RF1、MS8、Topas19/2、Oxy235、T45、GT73、RF2、RF3等為研究對象,建立的多重PCR檢測技術(shù)完全可滿足目前檢測和監(jiān)管的需求。主要研究內(nèi)容和結(jié)果如下:(1)轉(zhuǎn)基因水稻轉(zhuǎn)化體特異性多重定性PCR檢測技術(shù):針對轉(zhuǎn)基因水稻TT51-1、KMD1、KF6的旁側(cè)序列設(shè)計(jì)轉(zhuǎn)化體特異性引物,通過引物篩選,多重PCR反應(yīng)體系和反應(yīng)條件優(yōu)化,建立了轉(zhuǎn)基因水稻多重定性PCR檢測方法。通過擴(kuò)增轉(zhuǎn)基因水稻、轉(zhuǎn)基因大豆、轉(zhuǎn)基因玉米、轉(zhuǎn)基因油菜、轉(zhuǎn)基因棉花等不同作物來測試所選擇引物的特異性。結(jié)果表明,所建立的轉(zhuǎn)基因水稻多重定性PCR檢測方法具有較強(qiáng)的特異性,檢出限達(dá)0.1%。(2)轉(zhuǎn)基因油菜轉(zhuǎn)化體特異性多重定性PCR檢測技術(shù):針對轉(zhuǎn)基因油菜RF1、MS8、Topas19/2、Oxy235、T45、GT73、RF2、RF3 品系的旁側(cè)序列設(shè)計(jì)轉(zhuǎn)化體特異性引物,通過引物篩選,多重PCR反應(yīng)體系和反應(yīng)條件優(yōu)化,建立了轉(zhuǎn)基因油菜多重定性PCR檢測方法。經(jīng)對轉(zhuǎn)基因油菜、轉(zhuǎn)基因大豆、轉(zhuǎn)基因玉米、轉(zhuǎn)基因水稻、轉(zhuǎn)基因棉花等不同作物進(jìn)行PCR擴(kuò)增來測試所選擇的引物特異性。結(jié)果表明,建立的轉(zhuǎn)基因油菜多重定性PCR檢測方法具有較強(qiáng)的特異性,檢出限為0.05%。(3)轉(zhuǎn)基因油菜轉(zhuǎn)化體特異性多重定量PCR檢測技術(shù):針對轉(zhuǎn)基因油菜RF1、Topas19/2、Oxy235、RF3品系的旁側(cè)序列設(shè)計(jì)轉(zhuǎn)化體特異性引物和探針,通過引物和探針篩選,多重實(shí)時(shí)熒光定量PCR反應(yīng)體系和反應(yīng)條件優(yōu)化,利用相應(yīng)標(biāo)準(zhǔn)品梯度稀釋繪制標(biāo)準(zhǔn)曲線,確定所建立轉(zhuǎn)基因油菜多重實(shí)時(shí)熒光定量PCR的檢出限。隨后采用轉(zhuǎn)基因油菜、轉(zhuǎn)基因大豆、轉(zhuǎn)基因玉米、轉(zhuǎn)基因水稻、轉(zhuǎn)基因棉花等不同代表作物進(jìn)行PCR擴(kuò)增來測試所選擇的引物和探針的特異性。結(jié)果表明,所建立的轉(zhuǎn)基因油菜轉(zhuǎn)化體特異性多重定量PCR方法特異性強(qiáng)和靈敏性高,其檢出限為0.05%。
[Abstract]:With the increasing number and variety of genetically modified crops and their products, the potential risks caused by them have attracted much attention.GMO biosafety supervision is related to food safety, food safety and ecological safety. Countries and regions represented by the European Union have issued a series of laws and regulations to strengthen supervision.The detection of transgenic components is an important technical support and means for the safety supervision of transgenic organisms.In recent years, traditional PCR detection technology has been unable to meet the needs of simple, fast and efficient detection of genetically modified components.Because of its high specificity, high sensitivity, low cost, high throughput and saving experimental samples and drugs, multiplex PCR(Multiplex has been widely studied and applied in the field of transgenic component detection.In this paper, the transgenic rice TT51-1KMD1 + KF6 and the transgenic rape RF1MS-8 Topas19 / 2 Oxy235T45 T45 / GT73 RF2RF3 were used as the research objects. The established multiplex PCR detection technology can completely meet the needs of current detection and supervision.The multiplex PCR reaction system and reaction conditions were optimized, and a multiplex qualitative PCR detection method for transgenic rice was established.The specificity of the primers was tested by amplification of transgenic rice, transgenic soybean, transgenic corn, transgenic rapeseed, transgenic cotton and other crops.The results showed that the multiplex qualitative PCR detection method for transgenic rice had strong specificity.A multiplex qualitative PCR detection method for transgenic rapeseed was established.The PCR amplification of different crops such as transgenic rapeseed, transgenic soybean, transgenic corn, transgenic rice and transgenic cotton was carried out to test the specificity of the selected primers.The results showed that the multiplex qualitative PCR detection method for transgenic rapeseed had strong specificity.The reaction system and reaction conditions of multiplex real-time fluorescent quantitative PCR were optimized. The detection limit of multiplex real-time quantitative PCR of transgenic rape was determined by using the standard curve drawn by gradient dilution of the corresponding standard.Then the PCR amplification of different representative crops such as transgenic rape, transgenic soybean, transgenic corn, transgenic rice and transgenic cotton was carried out to test the specificity of the primers and probes selected.The results showed that the established transgenic rapeseed transformant specific multiplex quantitative PCR method had strong specificity and high sensitivity, and its detection limit was 0.05%.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S565.4
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本文編號：1722514