Construction And Application Of The Plasmid Standard Reference Molecule In Genetically Modified Maize

With the the rapid development of genetically modified organisms,their safety issue caused a heated debate and widespread concern all over the world.Countries around the world are working at developping relative laws and regulations,strengthening the management of the import and export of genetically modified plants and their products.To ensure the smooth implementation of labeling management regulation of genetically modified organisms(GMOs)in China,we carried out the study of the technology for the identification and quantification of GMOs,and two aspects was covered in this study:Firstly,construction of the positive standard reference molecule for the qualitative and quantitative PCR detection for genetically modified maize Bt11,Bt176,MON810 and MON863.In our current national standards,already known genetically modified products were used as positive control.With more and more testing work,demand for positive control is growing.Certified Reference Material(CRM) is quite important for GMOs identification and quantification.To resolve the problem that the present gene DNA preservation duration is short,and extracting too often consumes a certain amount of time and reagents,in this study, we constructed the positive standard reference molecule for qualitative and quantitative PCR detection for genetically modified maize Bt11,Bt176,MON810 and MON863.The event-specific sequences and endogenous gene were built on the different sites of the palsmid in two ways,and whether the two fragments were cloned into the pMD18-T vector was tested through enzyme cutting by two kinds of restriction endonuclease.Then we verified its applicability through the PCR.The results show that using the positive standard reference molecule were the same as the genetically modified genomic DNA samples.This indicated that the PCR results were reliable,the recombinant plasmid was available as a positive control for the genetically modified products testing,and the standard reference molecule existed as a circular DNA,with the advantages of simple extraction,easy preservation and using less.Secondly,produce standard curve with for GM maize positive standard reference molecule to determine the concentration of exogenous gene intransgenic plants.The work,in this article,was based on the obtained GM maize standard reference molecule pMD-ZBt11, pMD-ZBt176,pMD-ZM810 and pMD-ZM863,confirming the PCR detection sensitivity and limit(LOD).PCR results showed that the LOD of qualitative and quantitative PCR system is about 10 copies,besides,the test of the repeatability of standard curve proved its accuracy.The results show that the standard reference molecule is suitable for quantitative detection of genetically modified as certified Reference Material.