Construction of a Reference Plasmid for the Quantification of Genetically Modified Rice Kemingdao 2 and Uncertainty Evaluation

With the rapid increase in the number of biotech crops used for food production,consumers,researchers and governments are concerned about the health risks and environment safety posed by genetically modified(GM) crops and their derivatives.Reference plasmids are carriers of GM nucleic acid of the value and are provided for the value accuracy,comparability and effectiveness of realization of GM nucleic acid.Here,a novel reference plasmid for quantification of Kemingdao2(KMD2) was reported.Plasmidic DNA containing rice KMD2 DNA fragments is used as real-time PCR standard and positive PCR control.The pKMD2 plasmid was constructed based on pEASY-T3 that includes a 106 bp fragment of RBE4 which enables positive control PCRs,and a 137 bp GM constructed specific fragment of KMD2 for foreign gene quantification.The preparation,homogeneity,stability and commutability of pKMD2 were processed.To verify the correct number ratio between the RBE4 and constructed specific fragments in the pKMD2,the data was generated from sequencing by three independent laboratories and performed by quantitative real-time PCR for batch characterization,resulted 1.032.The uncertainty was evaluated by the sources of homogeneity,stability and batch characterization,resulted 0.032.In conclusion,this newly constructed plasmid is suitable for the practical detection and quantification of GM rice Kemingdao2 and feed products containing this DNA fragment.