Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

Theo W Prins,Ingrid M J Scholtens,Esther J Kok,Richard A Van Hoof

doi:10.1007/s00217-016-2761-6

Abstract

The import and use of genetically modified organisms (GMOs) is strictly regulated in the European Union. In order to maintain the legislation on GMOs, a genetic element screening is generally applied as a first step to detect authorised as well as unauthorised GMOs. Subsequent identification of GMOs that relate to the detected elements is performed by the application of event-specific detection methods. However, as the diversity of GMOs on the world market is increasing, there is an ongoing need for methods for additional informative screening elements. Genes that are increasingly applied in GMOs are cry3A (including variants mcry3A and eCry3.1Ab) conferring resistance to Bt toxins, and gat, detoxifying glyphosate. Novel TaqMan PCR detection methods for element cry3A and construct gat/T-pinII were developed to support the identification of maize MIR604, 98140, 5307, canola 61061 and 73496, and soybean 356043. Also, other unknown (unauthorised) GMOs containing cry3A and/or gat/T-pinII can potentially be detected. Specificity, efficiency and sensitivity of the methods were evaluated.

Highlights

In the European Union (EU), the import and use of genetically modified organisms (GMOs) is strictly regulated
Under Regulation (EC) No 619/2011
Eur Food Res Technol (2017) 243:481–488 allowed on the EU market, screening strategies targeting GMO elements and constructs are applied to limit the number of event-specific methods that need to be performed [14, 37, 43]

Summary

Introduction

In the European Union (EU), the import and use of genetically modified organisms (GMOs) is strictly regulated. Eur Food Res Technol (2017) 243:481–488 allowed on the EU market, screening strategies targeting GMO elements and constructs are applied to limit the number of event-specific methods that need to be performed [14, 37, 43]. Where the detection of promoter P-35S from Cauliflower mosaic virus (CaMV) or terminator T-nos from Rhizobium radiobacter was once sufficient to detect most authorised GMOs, there now is a tendency to use a wide variety of promoters, coding genes and terminators in more recent GMOs. the use of plant-derived GMO elements requires the use of construct-specific methods instead of element-specific methods to avoid false positives in feed samples containing multiple species. We describe the development of TaqMan PCR detection methods for the coding sequence cry3A and the construct gat/T-pinII to facilitate the detection of present and future GMOs containing these elements. We describe the newly developed methods and evaluate these in the light of established ENGL method performance requirements [25]

Materials and methods

Results and discussion

Conclusion

Compliance with ethical standards