|
With the rapid development of the commercialization of genetically modified crops, genetically modified crops have been deep into the lives, create huge benefits for the harmonious development of the social, economic, environmental. To turn the safety of genetically modified crops but there are still a lot of controversy, governments have also introduced a series of transgenic product management, rules and regulations, requirements to label on GM and its products is GMO, even GMO content restrictions. Currently, many without the labeling of genetically modified crops and their processed products to enter the Chinese market, There is an urgent need to lower the genetically modified genetically modified crops in the content of DNA extraction, concentration determination and studies of GMO detection methods, the establishment of a kinds of fast, easy and efficient detection method. The subject first corn seed material, improved CTAB method spin column (TIANGEN), spin column method (QIAGEN) beads (Promega) 4 genomic DNA extraction method and UV spectrophotometry, Qubit fluorescence method Second for the material to turn phytase maize, build phytase gene standard plasmid, establishing corn transit phytase gene PCR detection method; final design, Pico Green fluorescence spectrophotometry three kinds of DNA concentration determination method for the evaluation and analysis; and filter out the detection of the LAMP reaction primers corn transit phytase gene, the establishment of the maize transit the phytase gene LAMP quantitative detection methods, curve the LAMP detection standards build phytase gene. The key findings are as follows: (1) beads (Promega) is effective to obtain high purity, integrity, good genomic DNA, and reproducible, the extraction rate, 813.7 ng/100mg of the short extraction time may be in a hours completed; concentration determination of the genomic DNA, UV spectrophotometry, Qubit fluorescence method Pico Green fluorescence spectrophotometry relative error were 99.8%, 49.8%, 28.9%, and the Pico Green fluorescence spectrophotometry highest accuracy . (2) gene cloning, obtain the qualified standard of the phytase gene plasmid; established optimal reaction system and the reaction conditions of the PCR assay, the specificity and sensitivity of experiments showed that the PCR primers having good specificity and sensitivity for 1000 copies number, the the phytase gene PCR detection method of corn transit. (3) designed and screened for the LAMP detection phytase gene the primer group 1, to establish the optimal reaction system and the reaction conditions of the LAMP detected, the specificity and sensitivity of experiments show that the PCR primers have good specificity and sensitivity for 30 copy number, establishing corn transit phytase gene LAMP detection method; construct a the phytase gene the LAMP detection standard curve standard curve correlation coefficient R2 = 0.996, limit of detection of 60 of the phytase gene copy number.
|