转基因大豆、转基因马铃薯与阪崎肠杆菌的荧光PCR和数字PCR检测

发布时间：2018-04-28 08:09

本文选题：转基因品系 + 阪崎肠杆菌　；参考：《青岛大学》2017年硕士论文

【摘要】：随着转基因的安全性成为社会关注的热点,各国都相继出台了关于标识转基因产品的法规,而市场准入的关键就在于定性或者定量的转基因检测技术。本研究对转基因DAS-44406-6大豆品系、转基因AV43-6-G7马铃薯品系分别进行5′-RACE,测出两个品系外源基因片段和内源基因重组的边界序列,并按照边界特异性序列设计出相应的引物探针对,首次建立了转基因DAS-44406-6大豆品系和转基因AV43-6-G7马铃薯品系的实时荧光PCR和数字PCR检测方法。转基因DAS-44406-6大豆品系和转基因AV43-6-G7马铃薯品系的荧光PCR检测方法在模板DNA浓度为100 ng/反应时,检测低限均为0.01%的转基因含量;建立的转基因DAS-44406-6大豆品系和转基因AV43-6-G7马铃薯品系数字PCR检测方法绝对灵敏度能准确定量到模板DNA浓度分别为0.01 ng/μL和0.001 ng/μL的转基因样品。数字PCR方法相比于传统PCR检测方法具有简便快速、灵敏准确、可绝对定量等特点,这对于转基因品系的实际检测具有很大的应用价值。阪崎肠杆菌(Enterobacter sakazakii)可导致免疫力低下的新生儿神经系统紊乱如脑膜炎以及患菌血症、坏死性小肠结肠炎等疾病,婴幼儿配方奶粉是至今已知的最主要的感染途径。而检测阪崎肠杆菌的关键就在于实时荧光PCR和数字PCR检测方法。本研究首次建立了微滴式数字PCR检测阪崎肠杆菌的绝对定量检测方法。根据阪崎肠杆菌的特异性基因序列,设计引物和荧光探针,并进行筛选,和其它12种近缘的、食品中常见的其它菌种一起进行实时荧光PCR引物和探针的特异性以及检测低限实验,作为数字PCR的准备与参照。以阪崎肠杆菌基因组DNA制备绝对灵敏度与相对灵敏度的梯度稀释DNA进行PCR的定量检测,并确定和验证检测体系的稳定性、精密度、线性范围及检测低限等指标。本研究建立的检测阪崎肠杆菌的QX 200数字PCR检测方法定量检测低限为5×10-5 ng/μL,达到实时荧光PCR方法检测低限,同时在阪崎肠杆菌基因组浓度等于5×10-6 ng/μL时,仍可以检出病原菌,说明QX 200数字PCR检测方法灵敏度达到甚至超过实时荧光PCR方法。样品DNA模板浓度为5×10-3 ng/μL时,在阪崎肠杆菌基因组DNA相对含量区间为1.5625%-100%情况下具有较好的定量检出阪崎肠杆菌的能力;在样品模板浓度为5×10-3 ng/μL且阪崎肠杆菌在0.78125%-1.5625%时,也可以实现痕量阪崎肠杆菌样品的检出。首次建立的数字PCR检测方法具有较高的准确性和重复性、较短的检测时间,对快速检测出样品中的阪崎肠杆菌具有重要意义。
[Abstract]:With the safety of GM has become the focus of attention, many countries have issued laws and regulations on the identification of GMOs, and the key to market access is qualitative or quantitative transgenic detection technology. In this study, transgenic DAS-44406-6 soybean lines and transgenic AV43-6-G7 potato lines were carried out 5GRACE-RACE-RACE respectively, and the boundary sequence of exogenous gene fragment and endogenous gene recombination was detected, and the corresponding primer probe pairs were designed according to the boundary specific sequence. Real-time fluorescence PCR and digital PCR detection methods for transgenic DAS-44406-6 soybean and transgenic AV43-6-G7 potato strains were established for the first time. The fluorescence PCR detection method for transgenic DAS-44406-6 soybean lines and transgenic AV43-6-G7 potato lines was 0.01% when the template DNA concentration was 100 ng/. The absolute sensitivity of the established digital PCR detection method for transgenic DAS-44406-6 soybean strains and transgenic AV43-6-G7 potato strains can be accurately quantified to the transgenic samples with template DNA concentrations of 0.01 ng/ 渭 L and 0.001 ng/ 渭 L, respectively. Compared with the traditional PCR detection method, the digital PCR method has the advantages of simple, rapid, sensitive and accurate, and can be used in absolute quantitative analysis, which has great application value for the practical detection of transgenic lines. Enterobacter sakazakii) can lead to neurological disorders such as meningitis, bacteremia, necrotizing enterocolitis and so on. Infant formula is the most important way of infection. The key to detect Enterobacter sakazakii is real-time fluorescent PCR and digital PCR. In this study, an absolute quantitative method for detection of Enterobacter sakazakii by microdrop digital PCR was established for the first time. According to the specific gene sequence of Enterobacter sakazakii, primers and fluorescent probes were designed and screened. The specificity of real-time fluorescent PCR primers and probes and the detection of low limit test were carried out together with other common strains of food as preparation and reference for digital PCR. The absolute sensitivity and relative sensitivity gradient dilution DNA of Enterobacter sakazakii genomic DNA were used to quantitatively detect PCR, and the stability, precision, linear range and detection limit of the detection system were determined and verified. The quantitative detection limit of the QX200 digital PCR method for detection of Enterobacter sakazakii was 5 脳 10 ~ (-5) ng/ 渭 L, which reached the low detection limit by real-time fluorescence PCR method. At the same time, when the genomic concentration of Enterobacter sakazakii was equal to 5 脳 10 ~ (-6) ng/ 渭 L, the pathogen could still be detected. It shows that the sensitivity of QX200 digital PCR detection method is even higher than that of real time fluorescence PCR method. When the concentration of sample DNA template was 5 脳 10-3 ng/ 渭 L, the relative content of genomic DNA of Enterobacter sakazakii was 1.5625- 100%, and when the concentration of template was 5 脳 10-3 ng/ 渭 L and the concentration of Enterobacter sakazakii was 0.78125 and 1.5625%, Trace Enterobacter sakazakii samples can also be detected. The first digital PCR detection method has high accuracy, reproducibility and short detection time, which is of great significance for rapid detection of Enterobacter sakazakii in samples.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.1;S532;R378

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