A Rapeseed-Specific Gene, Acetyl-CoA Carboxylase, Can Be Used as a Reference for Qualitative and Real-Time Quantitative PCR Detection of Transgenes from Mixed Food Samples

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Abstract
Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA. Keywords: Brassica napus; rapeseed; canola; oilseed rape; colza; acetyl-coenzyme A carboxylase; Polymerase Chain Reaction; real-time quantitative PCR; GMO detection; reference endogenous gene