Research On Qualitative And Quantitative PCR Detection Technology Of Genetically Modified Soybean DP-305423

Genetically modified organisms and their product testing technology is an important guarantee of making system implementation.In this paper, we do qualitative and quantitative detection on the genetically modified soybean DP-305423specific DNA sequence,and established a set of complete for genetically modified soybean DP-305423qualitative and quantitative detection analysis method, the technical parameters can meet the demand of exogenous component testing. It has a wide significant to safeguard the interests of import and export trade,and promote the implementation of system.The study is dicided into thtee parts.The first part we make DP-305423soybean exogenous insert DNA fragments and plants of the genome sequence based basis for connection area,through specific detection primer design,primer screening,PCR detection reaction system and the reaction process optimization technology process,finally establish a DP-305423soybean qualitative PCR detection method,and validate the method of specificity,sensitivity,stability and repeatability.Using different genetically modified crops which we could collected as specific test object,the results show that the method can specific separate DP-305423and other genetically modified soybean、corn、canola、rice、and non-modified soybean.Using DP-305423and its receptor in accordance with the quality than mixed makes up to10%,5%,1%,0.5%,0.1%,0.05%six gradient diluted sample sensitivity tese,and the results show that the test method of sensitivity can achive0.05%,equivalent to about20soybean genome copy.We did stability and repeatability tests inl%,0.1%and0.05%DP-305423soybean,the result show that the test have good stability,high repeatability,and false negative rate is O.Comprehensive above the results,the study established DP-305423soybean specific PCR detection qualitative transformation body method has very good specificity and sensitivity,and other commercialized transgenic crops or the genetically modified soybean genome near genes without cross reaction.The second part, we clone the sequence of lectin gene and DP-305423soybean specific sequence to the commercial plasmid DNA on the carrier of the strategy, and construct the DP-305423standard molecular pMD-L305-2, and varify the qualitative and quantitative PCR test. Using0.1×TE solution dilute the plasmid standard molecular into gradient samples and have qualitative PCR test, the results show that the plasmid standard molecules of qualitative PCR detection sensitivity can reach up to10copy. Make diluted plasmid standard molecular solution in different temperature environment in stability testing, the results show that copy number concentration for103copy/μL and above the plasmid standard molecular has good stability, in4℃save15d,-20℃for more than30d can still expand the product steadily. Use lectin as soybean internal genes, by using of standard curve method to establish DP-305423soybean specific real-time fluorescence quantitative polymerase chain reaction (PCR), the results indicate that the plasmid standards established lectin gene and the molecular DP-305423soybean specific sequences of standard curve of the slope were3.583and3.536, correlation coefficient (R2) are more than0.999. Using known of quality score of DP-305423soybean quantitative PCR test sample experiment, the result shows that, based on the plasmid standard molecules samples DP-305423soybean composition content determination of value with the theoretical value average deviation is1.27%～22%, in line with international recognition range. Comprehensive above research results, the study of building up the plasmid standard molecular pMD-L305-2alternative DP-305423soybean matrix material, use it as the detective sample of DP-305423soybean specific PCR and real-time PCR quantitative fluorescence.The third part, construct a plasmid standard molecules with DP-305423hra gene sequence and lectin gene sequence, according to the plasmid standard molecular we build lectin gene and hra gene real-time PCR fluorescent quantitative standard curve, identified the copy number of DP-305423hra gene. The results showed that the slope of lectin gene and hra gene standard curve were-3.112and-3.235, correlation coefficient (R2) were0.9987and0.9992, standard deviation between0.00to0.01, using this method to determine DP-305423soybean genome hra gene for single copy. Research shows that, the real-time quantitative PCR technology which depended on the plasmid standard molecules can be applied to analysis the hra gene copy number of the DP-305423soybean.