Establishment Of A New Molecular Detection System For Genetically Modified Maize Seeds

With the rapid development of global transgenic technology.By 2020,there are 240 kinds of maize transformants in the world,and my country has independently developed and issued a total of 5 kinds of safety certificates.At present,the standard methods for transgenic detection in my country are mainly agarose gel electrophoresis and real-time fluorescent quantitative PCR.With the increase of genetically modified varieties,the existing detection methods has the disadvantages of low throughput,long time,high cost,and limited technical means.The difficulty of detection continues to increase.In order to solve the above situation,this article uses fluorescent capillary electrophoresis to establish a triple PCR that will screen quickly screen for genetically modified ingredients and a five-fold PCR that would lock domestic Maize transformants in one step.At the same time,the authenticity and genetic modification are combined to test,and the seed fingerprint information is screened at the same time.Genetically modified ingredients,to achieve the purpose of one-time double verification of Maize seeds.Using the most representative Maize transformant C0030.3.5 and Shuangkang 12-5transformant in China as the research materials,and set the 7 transgene content gradients,which were 0.01%,0.05%,0.1%,0.5%,1%,5%,and 10% respectively.A gradient of transgene content,using Maize internal standard ZSSIIb,Ca MV35 S promoter,T-NOS terminator,C0030.3.5,double antibody 12-5,a total of 5 pairs of primers,and then based on fluorescent capillary electrophoresis for single,triple,and five-fold PCR amplification increase.1.Establishing a fluorescent capillary electrophoresis system for detecting transgenes.Single-plex PCR was performed on 5 pairs of primers.Fluorescence capillary electrophoresis results showed that the detection limit of C0030.3.5 T-NOS primer and double antibody 12-5Ca MV35 S primer was 0.05%,which was higher than the two methods of agarose gel electrophoresis and q PCR The detection limit of 0.1%,the detection limit of the remaining primers in capillary electrophoresis is 0.1%,which is the same as the standard method.2.Establishing a triple PCR amplification system and verify it.ZSSIIb,Ca MV35 S and T-NOS were combined into triple PCR to amplify the C0030.3.5 Maize sample.The detection limit of triple PCR was 0.1%,and the detection limit of T-NOS primer was changed from 0.05%in single PCR to 0.1%.Using the TC1507,NK603,Mon810 Maize standard product verification system with genetically modified content of 0.5%,1%,and 2%,the amplification results are consistent with the facts,proving that the triple system can be used for rapid screening of genetically modified Maize seeds.3.Establishing a five-fold PCR amplification system and verify it.Combining 5 pairs of primers to amplify two Maize samples,the detection limit of five-fold PCR is 0.1%.The detection limit of T-NOS of C0030.3.5 and Ca MV35 S of double antibody 12-5 is changed from0.05% of single-plex PCR.Is 0.1%.Using the 100 Maize blind sample verification system mixed with C0030.3.5 and double antibody 12-5 samples,a total of 4 copies of C0030.3.5 were detected,2 double antibodies 12-5,and 13 copies contained genetically modified ingredients,which were in line with expectations.4.The combination of authenticity and GMO testing.The combination of ZSSIIb,Ca MV35 S,T-NOS and 8 pairs of SSR authenticity detection primers,using 11-fold PCR,based on a fluorescent capillary electrophoresis platform,can achieve simultaneous detection of authenticity and transgene.5.The above three detection platforms were compared.Comprehensive sensitivity,specificity,ease of use,throughput,time,cost and other 6 aspects,agarose gel electrophoresis is suitable for the qualitative detection of a small number of samples for genetic modification,q PCR can be used for relatively quantitative genetic detection,and fluorescent capillary electrophoresis is used for detection of large quantities.Genetically modified samples.This study is based on a fluorescent capillary electrophoresis platform to establish a multiple genetically modified detection system,which would integrate authenticity and genetically modified detection,providing a new detection program for the future genetically modified seed market,and has high application value and prospects in Maize seed genetically modified detection.