The Research On The Extraction And Concentration Determination Of Genome DNA And The Detection Methods Of Phytase Gene From Maize

With the rapid development of GM crops commercialization, GM crops have been deep into the lives and created great benefits for the harmony development of society, economy, and environment. However, there are still many controversial issues on the safety of GM crops. Governments have also introduced a series of management, rules and regulations in order to require labeling the GM crops and its products. Recently, many GM crops and its products have entered Chinese market without labeling, which makes it urgent and necessary to establish rapid, simple, efficient methods for the detection of the lower content of GM by the research of genome DNA extraction, concentration determination and transgenic ingredient detection.In this study, three aspects have been researched. Firstly, four genome DNA extraction methods, modified CTAB, Centrifuge column extraction method (TIANGEN), Centrifuge column extraction method (QIAGEN) and magnetic extraction method (Promega), and three DNA concentration determine methods, ultraviolet spectrophotometry, Qubit fluorescence and Pico Green fluorescence spectrophotometry have been applied to analyze and evaluate using the maize seeds. Secondly, standard plasmid with phytase gene has been constructed and the polymerase chain reaction (PCR) detection method of phytase gene in genetically modified maize has been established. Thirdly, LAMP reaction primers have been designed and selected, which are used for detecting phytase gene in genetically modified maize. Moreover, loop-mediated isothermal amplification (LAMP) assays for detection of phytase gene in genetically modified maize has been established and standard curve of LAMP detection for phytase gene has been constructed. The main results are as follows:(1) The magnetic extraction method (Promega) has high efficiency and good repeatability, requires short extraction and can produce genome DNA with integrality and high purity. Pico Green fluorescence spectrophotometry has the highest accuracy for determining the concentration of genome DNA.(2) The standard plasmid with phytase gene has been acquired by cloning. The reaction components and conditions of PCR have been optimized. The results of specificity and sensitivity assays indicated that the PCR primers had high specificity and the sensitivity could reach 1000 copies. Consequently, the polymerase chain reaction (PCR) detection method of phytase gene in genetically modified maize has been established. (3) A set of LAMP primer has been confirmed for the detection of phytase gene and the reaction components and conditions of LAMP have been optimized. The results of specificity and sensitivity assays indicated that the LAMP primers had high specificity and the sensitivity could reach 30 copies. So loop-mediated isothermal amplification (LAMP) assays for detection of phytase gene in genetically modified maize has been established. Furthermore, Standard curve of LAMP has been constructed for the detection of phytase gene, with a correlation coefficient of R2=0.996, and the detection limit has reached 60 copies.