Single laboratory method performance evaluation for the analysis of Roundup Ready® soy flour by qualitative and quantitative detection methods

Abstract

The Republic of Turkey has approved 7 soybean and 25 corn genetically modified (GM) events for animal feed use only and the biosafety legislation has banned the cultivation of GM crops and requires that all genetically modified organisms (GMOs), including imports, to be approved for use and further establishes a strict policy of testing for food, feed and seed potentially containing GMOs. For the GMO analysis, each laboratory should establish the verification on method performance criteria and calculation of measurement uncertainty. The aim of this study is to define the verification of qualitative and quantitative detection of Roundup Ready® soybean as a model for single laboratory verification in the context of the European Network of GMOs Laboratories guidance documents. First, two methods were used for the extraction of nucleic acids (DNA) and their extraction efficiencies were compared based on the quantity, purity, fragmentation state of DNA and inhibition in polymerase chain reaction (PCR). Second, a verification procedure of a real time PCR method for qualitative detection of cauliflower mosaic virus 35S promoter and Agrobacterium tumefaciens Tnos sequences in DNA samples extracted from certified reference materials and GM soy flour samples was performed. Last, the standard curves were prepared in order to explain verification of quantitative real-time PCR analysis by reaching the ideal value of -3.62 for lec reference gene and -3.40 for A. tumefaciens strain CP4 5-enolpyruvulshikimate-3-phosphate synthase (EPSPS) enzyme target gene. All method performance criteria for quantification (within-laboratory reproducibility standard deviation, relative standard deviation, uncertainty, bias, limit of detection, limit of quantification, and linearity) were met and thus the method in this study was verified. Finally, the document highlights a clear example for analysis of GMOs in food and feed samples, and points out the need for interlaboratory studies at the national and international level.