The Study On The Detection Of Genetically Modified In Foods Using Loop-mediated Isothermal Amplification

With the help of modern molecular technology,, exogenous genes could be transplanted into genetically modified foods so that genetic material could be modified and some characters such as appearance, nutrition and consumption quality could be improved. Genetically modified foods or biotech foods are foods derived from genetically modified organisms.In this paper genetically modified foods mean foods of plants origin. Since the first commercialization of genetically modified tomato in1993, a94-fold increase in hectarage from1.7million hectares in1996to160million hectares in2011has been reached (almost the same size as China or America). Of the29countries planting biotech crops in2011, it is noteworthy that19were developing countries and10were industrial. A total of16.7million farmers grew biotech crops, generating$1.5billion output. The addition of about60countries of the global endorsement of use (cultivation or imports) more than30kinds of genetically modified crop products,60%of world’s population,,4billion people, live in the29countries planting biotech crops. These data indicate that genetically modified crop has gradually scaled. Genetically modified food was not far away from the daily lives.The traits of the species, the impact on environment, and the effects on human beings are not clear. Not only laws and regulations for monitoring have to be passed but also a series of testing to determine the GMO standards should be developed. The detection of genetically modified products could be mainly divided into two categories:protein level and molecular level detection. However, the GMO, its multi-channel program processing, the vast majority of protein denaturation, destruction, lower detection rate. Molecular level detection is more widely used in a variety of the scientific researches and practical applications.There are common molecular detection methods:(1) PCR technology:it is divided into ordinary qualitative PCR, nested PCR, and fluorescence quantitative PCR;(2) gene chip technology;(3) other new technologies:the RAPD technique, the AFLP technique, SSR and ISSR. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method developed by Notomi et al. It is characterized by the use of4different primers specifically designed to recognize6distinct regions on the target gene(upstream internal primers FIP, downstream internal primers BIP, upstream external primers F3and downstream primers B3) and the reaction process proceeds at a constant temperature(about65℃)using a DNA polymerase with strand displacement activity. It provides high amplification efficiency, with DNA greatly amplified in less than60min. Therefore, it has the potential to replace PCR because of its simplicity, rapidity, specificity, and cost-effectiveness.LAMP detection was applied to detect exogenous and endogenous reference genes four strains, including genetically modified soybeans GTS40-3-2, GM maize of MON810genetically modified maize BT176transfer of transgenic rice TT51-1.The study is divided into three parts:Find the relevant literature, genetically modified soybeans GTS40-3-2outside source insert gene CP4EPSPS for the object of study, to explore the reaction system of LAMP detection technology and the value of the parameter. Concluded through a series of experiments to explore the experimental conditions and parameters:The LAMP reaction was carried out in a25μL total reaction mixture volume with containing betaine,2.0mmol/L Mg2+,0.8μL the Bst DNA polymerase,0.8mmol/L dNTPs c primer concentration inside and outside the ratio of1/8, the temperature was65℃, time60min, the LAMP reaction system is relatively stable, clear bands of amplification products. We designed the primers for the gene which be detected.GTS40-3-2、MON810、 BT176and TT51-1positive standard as a template, respectively extracted genomic DNA. Then, F3/B3as primers oBTained the target gene fragment by PCR amplification. Insert it into the pMD-18T connection vector by T4DNA ligase. Positive recombinant plasmids were identified by plasmid PCR, while, DNA sequencing determined sequence fidelity.The results show that the corresponding genes’clones were successes, and they were correctly inserted into pMD-18T connection vector cloning sites. Standard plasmid molecules in accordance with the dilution of the way followed by preparation2000000copies/μl200000copies/μl,20000copies/μl,2000copies/μl,200copies/μl,20copies/μl,2copies/ul seven the gradient of the standard solution for LAMP detection. Determine the detection sensitivity and minimum detection threshold for each gene. The target fragment was digested by restriction enzyme to determine the accuracy of the detection technology. Amplify the different products with the same primers which can be used to determine the specificity of this detection method. And the two methods, Fluorescence qualitative PCR and SYBR Green Ⅰ method, were used to detect the experimental results. Finally, it was used to detect the actual processing products. The LAMP detection of each gene in addition to the minimum detectable value of transfer of genetically modified rice TT51-1strain-specific sequences:200copies/ul minimum threshold of detection by the remaining genes were less than200copies/μl. Restriction analysis shows the digested fragment size and theoretical fragment size is basically the same. Series of experiments to prove the feasibility of LAMP detection method, accuracy, repeatability, sensitivity and specificity are relatively good. It can be used for the detection of genetically modified products and processed products.In this study, we determined the species and the names of the gene by accessing the data of related articles, standard species and gene lists from Quarantine Bureau. Softwares such as Explorer4.0and premier6.0were used for designing primers of gene target fragment to be amplified.. A series of experiments to determine the optimal reaction conditions of the LAMP reaction. Construct standard plasmid molecules to determine the LAMP detection method to detect the sensitivity of each gene, and finally applied to the detection of agricultural products and processed products. Initially verify the feasibility of LAMP detection method for the detection of genetically modified crops and their processed products. These lay the foundation for further research for the future of LAMP detection method to detect genetically modified.