Establishment and Validation of Reference Genes of Brassica napus L. for Digital PCR Detection of Genetically Modified Canola

Abstract

As an effective tool for genetically modified organism (GMO) quantification in complex matrices, digital PCR (dPCR) has been widely used for the quantification of genetically modified (GM) canola events; however, little is known about the quantification of GM canola events using endogenous reference gene (ERG) characteristics by dPCR. To calculate and quantify the content of GM canola using endogenous reference gene (ERG) characteristics, the suitability of several ERGs of canola, such as cruciferin A (CruA), acetyl-CoA carboxylase (BnAcc), phosphoenolpyruvate carboxylase (PEP), cruciferin storage (BnC1), oleoyl hydrolase (Fat(A)), and high-mobility-group protein I/Y (HMG-I/Y), was investigated by droplet dPCR. BnAcc and BnC1 were more specific and stable in copy number in the genome of Brassica napus L. than the other genes. By performing intra-laboratory validation of the suitability of ERG characteristics for the quantification of GM canola events, the ddPCR methods for BnAcc and BnC1 were comprehensively demonstrated in dPCR assays. The methods could provide technical support for GM labeling regulations.