基于DNA恒温扩增技术的转基因作物快速检测方法研究

发布时间：2018-06-30 17:35

  本文选题：转基因 + 恒温扩增　； 参考：《浙江大学》2016年博士论文

【摘要】：转基因作物的推广和商业化种植在带给人们巨大经济效益的同时,其潜在风险却也饱受争议,快速准确地检测转基因具有十分重要的意义,事关国际政治和经济利益。传统基于外源蛋白分析的检测方法具有明显的局限性,而基于外源核酸分析的检测方法在对操作人员有较高要求的同时又受制于专业、昂贵、体积庞大的仪器设备。为了开发简单、快速、便携基于核酸分析的转基因检测方法,本课题以抗虫转基因水稻华恢1号、科丰6号和克螟稻1号为研究材料,以交叉引物恒温扩增技术(CPA)和环介导恒温扩增技术(LAMP)为扩增方法,以核酸扩增过程中的副产物H+和磷酸根为检测对象,分别建立了电化学无标记的核酸扩增检测方法和新型可视化核酸恒温扩增方法,并在以上研究基础上提出了基于逻辑判断的转基因具体品系的快速鉴定策略,开发出纸上可视化点阵列鉴定转基因品系的方法。本文的主要内容和研究结果如下：1)针对转基因通用元件T-nos的基因序列建立了用于转基因作物快速筛选的CPA体系,设计了6条引物,包括2条外引物、2条交叉引物和2条检测引物,特异性识别'T-nos序列的8个区域；通过使用SYTO 9作为荧光染料实现了对扩增反应的实时监测,考察了该体系的特异性和检测灵敏度,并与传统实时荧光PCR的检测效果进行了对比,结果表明：所建立的CPA体系能够特异性检测转基因农作物中的nos基因；其检测限约为103拷贝水稻基因组DNA,相比传统实时荧光PCR检测,能够达到相同的检测限但需要更短的时间(节约10 min左右)；除此之外,能够检测至少0.5%掺杂的实际样本,足以满足国际上大部分国家和地区对转基因检测的需求。综上所述,CPA体系能够作为快速、简便的核酸扩增方法取代传统的实时荧光PCR,表现为需要更短的时间、更简单的反应仪器。2)以核酸扩增过程中的副产物H+为检测对象,建立了基于pH变化检测的电化学无标记核酸扩增检测方法,搭建了实时分析和终点检测两种形式的检测平台,结果表明：最适合用于pH检测的CPA体系(pH-CPA)的组成为50mMK+,15mM NH4+,3 mM的KOH添加；使用商业化pH计可以对pH-CPA扩增进行实时监测,将扩增过程中的pH变化最快的点作为pH-CPA反应的信号阈值,通过对扩增过程中得到pH变化曲线进行求导,得到曲线的局部最小值即为pH-CPA的信号阈值,信号阈值所对应的时间即为"Time to threshold",用tth表示；利用tth分析,pH-CPA扩增方法表现出可靠的定量分析性能,10倍差异的模板,tth大约差3；相比实时荧光检测的方法,该电化学检测方法节省大约20%的时间；此外,通过使用可抛弃的pH敏感的丝网印刷微电极,初始模板量为103拷贝基因组DNA的阳性样本扩增前后峰值电位差△Ep发生28.3 mV的变化,对应为0.38个pH,与使用商业化pH计实时分析得到的结果大致相同(约0.35 pH)。综上可知,以溶液中的H+浓度的变化为检测对象能够实现对核酸扩增反应实时或是终点的电化学检测,无需庞大的仪器设备,与恒温扩增技术结合应用,使得核酸分析过程更便携、简单易用,由于电化学检测本身具有易于集成化的特性使得该方法具有更好的应用前景。3)通过检测核酸扩增过程中的Pi信号的产生,建立了基于Pi分析的可视化核酸恒温扩增检测方法,详细系统地考察了该方法应用于不同扩增体系的可行性,并设计了配套的可抛弃的防污染装置,建立了完整的快速、便携、可视化核酸扩增检测体系,结果表明：核酸扩增条件下非扩增特异性Pi产生的唯一来源是dNTP分解,影响dNTP分解最主要的因素是反应温度条件,传统PCR(加热／冷却热循环,40 cycles)条件下约13%的dNTP分解产生Pi,而恒温CPA扩增条件下(63℃,1 h)则仅有2%的dNTP发生分解；在扩增特异性Pi信号产生情况的研究中,经过PPase处理的CPA阳性样本约产生2.86 mM的Pi,是不经过PPase处理的阳性样本产生量的36倍,阴性样本产生量的53倍(经过PPase处理),而PCR体系阳性样本(经过PPase处理)则产生约0.5 mM的Pi,相应阴性样本(经过PPase处理)产生Pi0.15 mM；通过对dNTP利用率的计算可以得到CPA扩增体系中有大约87.5%的‘dNTPs被用于扩增,将近3.36%发生非扩增特异性降解；而PCR扩增体系则仅有22.3%的dNTPs被用于扩增反应,18.6%的dNTPs降解;使用该可视化方法检测CPA,仅需扩增15 min即可完成对103拷贝基因组DNA的检测,相比第3章基于pH变化的电化学检测核酸扩增的方法节约将近50%的时间；通过考察该方法在LAMP扩增中的应用,得知该方法并不局限于对CPA扩增体系的检测,具有能够应用于其他种类恒温扩增体系的巨大潜力。综上可知,以溶液中的Pi信号的产生为检测对象能够非常直接、简便地对核酸扩增结果进行判断,可视化信号非常明显、反应速度快,更大程度地发挥了恒温扩增用于现场、资源匮乏条件下进行核酸分析的优势。4)建立了基于逻辑判断的转基因品系鉴定方法,以国内3种常见抗虫转基因水稻(华恢1号、科丰6号和克螟稻1号)为研究对象,筛选出能够有效获取该组对象基因转化详细信息的基因组合,以LAMP为恒温扩增方法示例,通过Mo-Sb-Vc体系对结果显色,阳性结果表现为蓝色计作“1”,阴性结果显示为无色计作“0”,根据样品检测结果得到的类似“1101”的结果进行逻辑判断,最终鉴定出转基因具体品系,结果表明,同步检测sps、nos、 CaMV35S和crylAc基因需要扩增约35min,转基因掺杂度低至0.5%的样品仍能产生明显的颜色变化,检测结果基本不受深加工工艺过程的影响；使用Whatman 1级Chr层析纸作为基质,使用磷酸显色试剂对其进行预处理,根据阵列中显色得到的蓝色／无色斑点的数量及顺序即可鉴定出转基因样本的具体品系。综上所述,我们建立了一种新型的多基因分析策略用于抗虫转基因品系鉴定,具有信息量丰富、快速、简单易用、易于更新的特点,为多目标分析提供新思路。
[Abstract]:In order to develop a simple , rapid and portable method for the detection of transgenic lines , six primers were designed , including two external primers , two cross primers and two detection primers , which specifically identified 8 regions of the ' T - nos sequence .
By using SYTO 9 as the fluorescent dye , real - time monitoring of the amplification reaction was realized , the specificity and the detection sensitivity of the system were investigated , and compared with the traditional real - time fluorescence PCR detection results , the results showed that the established CPA system can specifically detect the nos gene in the transgenic crops ;
the detection limit is about 103 copies of rice genome DNA , compared with the traditional real - time fluorescence PCR detection , the detection limit can reach the same detection limit but shorter time ( about 10 minutes is saved ) ;
In conclusion , the CPA system can be used as a rapid and simple nucleic acid amplification method to replace the traditional real - time fluorescence PCR . In conclusion , the CPA system can be used as a rapid and simple nucleic acid amplification method to replace the traditional real - time fluorescence PCR .
A commercial pH meter can be used for real - time monitoring of pH - CPA amplification , and the pH value in the amplification process is taken as a signal threshold of the pH - CPA reaction , and a pH change curve is obtained through the amplification process to obtain a local minimum value of the curve , namely , the signal threshold value of the pH - CPA , the time corresponding to the signal threshold is " Time to threshold " , and is expressed by tth ;
Using tth analysis , the pH - CPA amplification method showed reliable quantitative analysis performance , 10 - fold difference template , tth about 3 ;
Compared with the real - time fluorescence detection method , the electrochemical detection method saves about 20 percent of time ;
In addition , by using a disposable pH - sensitive screen printing microelectrode , the peak potential difference between the pre - and post - amplification peak potential difference of 103 copies of genomic DNA , which corresponds to 0.38 pH , was approximately the same as the results obtained in real - time analysis using commercial pH meter ( about 0.35 pH ) . It is known that the detection target can realize the electrochemical detection of real - time or end - point of nucleic acid amplification reaction by the change of concentration of H + in solution . The method is more portable and easy to use because electrochemical detection itself has the characteristics of easy integration . The results show that the unique source of non - amplification specific Pi in nucleic acid amplification is the reaction temperature condition .
In the study of the generation of the amplified specific Pi signal , the CPA positive sample treated by PPase was about 2.86 mM Pi , 36 times the amount of positive sample that was not treated by PPase , 53 times the amount of negative sample ( treated by PPase ) , whereas the PCR system - positive sample ( treated with PPase ) produced about 0.5 mM Pi , and the corresponding negative sample ( treated with PPase ) produced Pi0 . 15 mM ;
The results show that the method is not limited to the detection of nucleic acid amplification system . The results show that the method is not limited to the detection of nucleic acid amplification system . The results show that the method is not limited to the detection of nucleic acid amplification system .
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S188
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本文编号：2086305