Study On Degradation And Detection Of Exogenous Genes In Transgenic Soybean And Rice By Key Factors In Food Processing

Nowadays,genetically modified crops are widely concerned,the safety of genetically modified food has always been a controversial issue.The detection of transgenic ingredientsat present is mainly based on DNA-based PCR technology.But,it is difficult to detect transgenic ingredients in processed food products,because processed food products have undergone a series of processing processes,their DNA degradation occurs to varying degrees,and false-negative test results are easily caused.The main research contents and results of this paper are as follows:1.Recovery of small fragments of DNAIn this experiment,six commonly used methods for DNA Extraction were compared,and it was determined that silicon beads in QIAEX II Gel Extraction Kit had the best effect in recovering small fragments of DNA.Then,the experimental conditions for silicon beads to recover small fragments of DNA were further optimized,and the best method for small fragments of DNA was finally determined.Finally,the optimized silicon beads and CTAB method were used to extract the DNA of transgenic soybean after high temperature treatment.The results show that the silicon bead method has the best ability to recover small fragments of DNA.2.Effects of different temperature and acid-base treatment on detection of transgenic soybeanThis study explores the different temperature and acid-base treatment,DNA test results of different fragment sizes in the 4 commonly used screening elements of transgenic soybean NOS,CaMV35 S,EPSPS and BT.And compared with results of national standard《NY/T 675-2003 transgenic plants and its product testing soybean qualitative polymerase chain reaction(PCR)》.The experimental results show that genetically modified soybean DNA is more sensitive to temperature,the higher the temperature of the processing,the longer the time,the greater the degree of DNA degradation.In addition,in the detection of transgenic ingredients,the target fragment with a small band is easier to be detected,while when the detection target band is large,false negative results are easily caused.3.Study the degradation rule of transgenic soybean DNA by temperature based on digital PCR technologyThe experiment further explored the law of gene degradation in transgenic soybean after temperature treatment.The degradation rules of NOS,CaMV35 S,EPSPS and BT genes were investigated by using digital PCR technology.The results showed that temperature can cause degradation of the four screening elements to varying degrees,and the higher the temperature,the greater the degradation degree.Meanwhile,among the four elements,CaMV35 S gene is the most sensitive to temperature,while EPSPS gene is the least sensitive to temperature treatment.4.Different PCR techniques were used to detect transgenic rice tt51-1 in food processingTo assess the effects of food processing on the detection and quantification of transgenic rice TT51-1 in processed food by polymerase chain reaction(PCR)technology,the presence of TT51-1 components in rice crackers at different processing stages was monitored by conventional PCR,quantitative real-time PCR(qPCR),and droplet digital PCR(ddPCR)using standard or validated primers and probes.For conventional PCR,the relative longer amplification targets,such as Bt gene(301bp)and event-specific target(274bp),were hard detected in baked,fried or microwaved samples.For qPCR,amplification fluorescence signal was detected in boiled,dried,baked,and microwaved samples,but it was difficult to observe the fluorescence signal in fried samples.Conventional PCR with the same primers that were employed in qPCR was used to detect corresponding shorter targets in all samples.These conventional PCR results were mainly consistent with the results of qPCR.The results indicated that food processing directly affects the detection of transgenic components,and suggested that relatively shorter fragments should be selected as the amplified targets for this type of analysis.The qPCR and duplex ddPCR methods were established for quantifying TT51-1.The results of an orthogonal experiment indicated that the optimal conditions for TT51-1/PLD duplex ddPCR were 500/250 nM of primers/probe combined with 58°C annealing temperature.Both methods were feasible for quantitative detection of TT51-1 in processed samples,with duplex ddPCR being a more attractive method for detecting transgenic components in processed food due to its stability,accuracy,PCR inhibitor resistance,and the lack of a need for reference materials.In summary,silicon bead extraction and recovery of small fragments of DNA is the best among the six common methods for DNA recovery.In the process of food processing,both temperature and pH value can cause DNA degradation,and temperature has a greater impact.Therefore,shorter target segments should be selected for detection,and digital PCR with more stable and reliable detection results should be selected.