A new dual plasmid calibrator for the quantification of the construct specific GM canola Oxy-235 with duplex real-time PCR

Abstract

To overcome the difficulties of obtaining the Certified Reference Material (CRM) and according to the key documents of the European Union Reference Laboratory (EU-RL), a new standard reference molecule containing the construct specific of the canola event Oxy-235 (3′-junction Nitrilase/Tnos) and the canola endogenous reference gene (acety-CoA-carboxylase) was constructed and used for duplex real-time quantitative analysis. The limits of detection (LOD) were less than 5 Haploid Genome Copy (HGC) and the limits of quantification (LOQ) were about 10 HGC. Furthermore, mixed GM and non-GM canola samples were analysed with duplex QRT-PCR to evaluate the performance criteria as required for validation procedures in the EU-RL, namely, the precision and the accuracy. The accuracy expressed as bias ranged from 2% to 10% and the precision (repeatability and reproducibility) expressed as the RSDr and RSDR was from 2.2 to 5.12 and 2.15 to 5.46 respectively. All these indicated that the developed construct specific method and the reference molecule are suitable for the identification and the quantification of the canola event Oxy-235.