Study On Quantitative Detection Of Genetically Modified Components In Foods

With the extensive planting of genetically modified (GM) crops worldwide, food safety and environmental security issue has been subject to the attention of consumers, governments and agency personnel. Many countries and regions have developed safety regulations, labeling system, and measurement technology of GM foods. The demand of positive control is also increasing with it widely used for GM foos detection. The traditional standard reference molecules are usually constructed by genetically modified materials and their corresponding non-genetically modified materials according to a certain proportion of the quality of preparation, to obtain a series of mass fraction of calibrators. However, many GM strains of calibrator samples are difficult to obtain due to the strict control of GM standard products in our country. Because of the advantages of plasmid standards containing endogenous genes and exogenous genes in terms of long-term stability, less dosage, easy preservation, and easy preparation, constructing plasmid DNA calibrators as positive control materials has become the research focus of new positive material in recent years. Currently, the detection level of GM components in foods has improved from the qualitative to the quantitative. There is a large defect in traditional quantitative PCR as it starts to quantitative test when reached amplification plateau but not eliminate the quantitative error caused by the differences of each target gene amplification efficiency. DNA concentration is proportional to the initial amount of target DNA in the PCR reaction if each cycle of PCR amplification efficiency of the reaction is consistent. However, amplification products were non-exponential amplification with unknown reaction rate since amplification efficiency is changing between different reactions, or different cycles in the same reaction, especially the late stage in the PCR cycling reaction. In order to get the highest detection sensitivity, terminal quantitative based on common PCR is often applied, when the amplification product reached maximum (plateau phase). But it is difficult to determine the relationship between the concentration of the end products and the initial concentration of the target gene for the consumption of reagents and polymerases gradual inactivation. In recent years, real-time quantitative PCR, integrates advantages of PCR and probe hybridization technology, can quantify DNA template copies accurately via detecting changes of the fluorescence signal in the PCR process, and its detection sensitivity, which is2pg transgenic DNA per gram of sample, is about100times higher than the conventional PCR, as well as it can quantify deep-processed products and mixed samples. Real-time quantitative PCR reaction system could real-time monitor the entire reaction changes with the reaction process compared to the end of measurement methods. The volume of amplification products and the release of fluorescent signal are associated, but also a certain proportion in Real-time quantitative PCR. The fluorescence signal increases proportionally with amplification products increase in each cycle of PCR. Recording the amount of fluorescence signal released in each cycle to monitor the amplification reaction of entire exponential phase.This paper aims to develop the plasmid molecules of several common genetically modified crops, and provides a set of simple, accurate, efficient GM foods quantitative detection system to solve the problems of lack of positive standard and complexity of detection for deep-processing products, as well as poor credibility. These provide a technology platform for quantitative testing standardization of detecting GM components in major crops, a technical assurance to establish a practical detection system of the deep-processing products and the effective implementation of the GMO labeling system, and a technical support of GMO identification, the detection of environmental safety and using safety and long-term tracking and monitoring, surveillance and security risks of the forecast, early warning. In this paper, we mainly focus on the method research using PCR detection, and collected genetically modified materials by reference to the latest National Standard, Announcement of the Ministry of Agriculture (MOA), Entry-Exit Inspection and Quarantine Industry Standard of our country and European Union (EU) Standard. Five major species of13strains based on qualitative and quantitative methods were established, including10strains of GM maize (NK603, Btll,59122, of MON810, T25, GA21, MIR604, MON863, TC1507, Bt176) and each of GM soybean event (GTS40-3-2), GM rape event (RT73), GM rice event (TT51-1), GM sugar beet event (H7-1). These species cover the main import and export transgenic plants which have approved and soon to be approved in our country. The target sequences consist of endogenous genes and event-specific sequences, which approximately include20common genes or sequences. In the initial research stage, we tested CRMs to verify and optimize reaction condition and reaction system to the best, then applied these methods to detect transgenic plants of seeds, feed, food and deep processing products after getting a stable, accurate, efficient detection methods. Meanwhile, amounts of nine new standard plasmid molecules from13strains had been constructed in the article, and the sensitivity, stability and accuracy indicators in real-time quantitative PCR detection system had been further identified. We developed quantitative PCR assay for detection of13GM plant lines and analyzed event-specific sequences of these trains using Taqman probes with effectively applied to the detection of early processing and deep processing genetically modified foods.In the part of development of the plasmid calibrators,13reference molecules of strains, including five main species (genetically modified soybeans, crn, rapeseed, rice, sugar beet), were successfully constructed using overlap extension PCR. The technology is different from the traditional digestion connections, because it does not require restriction enzymes and ligase to DNA fragments but can fast connection in vitro. To minimize nucleotide mismatches, overlap extension PCR reactions generally should use high-fidelity DNA polymerase from some researchers suggest, but it could be done by means of a non-fidelity Taq polymerase, the amplification products are automatically added the base A in the3’end without3’â†'5’exonuclease enzyme activity, to complete the overlap extension PCR in our experiment, and it is also very stable and non-fidelity enzyme amplification which can be seen from the experimental results. During the experiment, the comparison of the fidelity and non-fidelity enzyme using overlap extension PCR experiments have been implemented, we found that the amplification efficiency were almost no substantial difference when fragment within1000bp by these two enzymes. In addition, two methods were designed to the primers for overlap extension PCR:firstly, the complementary base sequences, nothing to do with the primers, were adding to the reverse primers of endogenous gene and forward primers of exogenous gene. From this way, the recombinant DNA contains the "extra" fragment connected to the middle. Secondly, the base sequences, complementary to the primers, were adding to the reverse primers of endogenous gene and forward primers of exogenous gene, that is, the overlapping sequence added on endogenous gene reverse primer is the complementary sequence of exogenous gene forward primer and the overlapping sequence added on exogenous gene forward primer is the complementary sequence of endogenous gene reverse primer. The recombinant DNA fragment obtained in this way is length of both amplified fragment but without the "extra" fragment. The experimental results showed that the second approach, the design of primers to amplify recombinant DNA, is better than the first approach.Standard reference molecules we constructed were recombinant plasmid molecules containing one or more foreign sources genes and event-specific sequences, as well as the specific fragment of the endogenous gene identified. The strategy of constructing standard reference molecules is based on detection policy of genetically modified organisms. The reference molecules constructed in this study contain the endogenous gene and its event-specific sequence, which there are five plasmids (pMD-1G, pMD-a5, pMD-FR, pMD-ST and pMD-GH) containing an event-specific sequences and an endogenous gene sequence, and fours (pMD-aNB, pMD-aMB, pMD-aGM and pMD-aMT) containing two event-specific sequence and an endogenous gene sequence. The event-specific detection method has a high-specificity and accuracy, and more applicable to construct standard curves of quantitative PCR detection required for the connection area of the carrier with specific exogenous inserted and plant genome sequence, which is a single copy sequence and is more accurate than other exogenous gene copy number in genomic DNA standard.In the study of quantitative PCR detection method, in order to better evaluate the feasibility of real-time fluorescence quantitative PCR detection system, we further analyzed its sensitivity, stability, accuracy indicators and chosen20000copies/μl,2000copies/μl,200copies/μl,20copies/μl,2copies/μl of five concentrations to determine LOD and LOQ of quantitative PCR, which values were2copies and20copies, respectively. At the same time, we took GM soybean for instance to test stability of pMD-1G reference molecule, the SD values and the RSD values are within the normal. All of the above results indicated that the pMD-1G could be successfully used as standard material for quantitation of GM products. Under ideal conditions, the amplification efficiency of the different genes in the reference molecules is consistent, that is, the copies ratio of foreign gene and endogenous gene is100%, but other factors often lead to inconsistent amplification efficiency in the actual testing. In this study, we put the standard sample of1%mass fractions of GM soybean as the detected object, and calculated the average Cf value (0.94), which SD and RSD were0.11and11.57%, respectively. According to this, it could be inferred that the pMD-1G reference molecular amplification in quantitative PCR reaction system almost simultaneously. Especially,5%,2%,1%,0.5%mass fraction of the standard samples were tested to measure reference molecule for quantitative detection accuracy, the results showed that the differences between the actual measured GMO contents with true values were increasing with samples of genetically modified content increased, from which the detection bias (bias%) of15%and2%of the samples less than5%, and the value could be a larger deviation when low content of genetically modified organisms be deteced.After a comprehensive assessment of the various indicators of the plasmid molecules, we constructed the standard curve of13strains using Taqman probe method. The sample amplification efficiency (Eff%) of each group were from 95.007%to118.438%within the normal range, the square regression coefficients (R2) were greater than/equal to0.990, and each group of samples of three parallel experiments were highly repetitive. Good linearity between copy number and fluorescence values (Ct) visualized in the calibration curves indicated that these13real-time PCR assays combined with reference molecule established in this study were well suited for quantitative measurements. In addition, according to2692bp in length of the pMD-18T vector and13strains of genetic group of DNA length, we diluted into2000000copies/μl,200000copies/μl,20000copies/μl,2000copies/μl,200copies/μl,20copies/μl standard solution of six gradient using reference molecule, respectively. Thereby, the standard curve can be accurate quantification of100ng0.1%-100%transgenic samples of genomic DNA to meet the various national GMO labeling system (0.9%-5%) required. Based on these, we studied a quantitative analysis of plant-derived foods, including raw, primary processing and deep processing of a total of11kinds of food, which the minimum quantitative value was1.27%. These results proved that our research can be successfully applied to genetically modified food using quantitative detection method.