Establishment and in-house validation of simplex and duplex PCR methods for event-specific detection of maize SYN-E3272-5 using a new reference molecule

Abstract

Despite rapid developments in the detection techniques for genetically modified organisms (GMOs), the event-specific PCR method with high specificity is still the most used technique. In this study, event-specific simplex and duplex qualitative and quantitative detection systems were developed targeting the 3′ insertion site of GM maize SYN-E3272-5 (3272) construct. A reference molecule p3272 was constructed to act as positive control and as calibrator for quantitative analysis. The LOD for simplex and duplex qualitative PCR assays was 10 copies of p3272 control DNA. LOD and the LOQ for simplex and duplex quantitative PCR assays were 10 and 25 copies of p3272 DNA, respectively. Furthermore, four practical GM maize samples were quantified using the established simplex and duplex quantitative PCR systems by in-house validation. Results from five operators showed that the bias ranged from 3.44 to 17.24% in the simplex system and from 0.42 to 16.06% in the duplex system, respectively. These results demonstrated that the established event-specific simplex and duplex qualitative and quantitative PCR systems combined with the reference molecule p3272 are suitable for the detection of GM maize 3272 and its derived products.