Event-Specific Plasmid Standards and Real-Time PCR Methods for Transgenic Bt11, Bt176, and GA21 Maize and Transgenic GT73 Canola

• Published in: Journal of agricultural and food chemistry Vol. 53; no. 8; pp. 3041 - 3052
• Main Authors: Taverniers, Isabel, Windels, Pieter, Vaïtilingom, Marc, Milcamps, Anne, Van Bockstaele, Erik, Van den Eede, Guy, De Loose, Marc
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 20.04.2005
• Subjects: Bacillus thuringiensis; bar gene; Base Sequence; Biological and medical sciences; Brassica napus; Brassica rapa - genetics; canola; Cereal and baking product industries; corn; cry gene; crystal proteins; DNA primers; DNA probes; DNA, Plant - chemistry; EPSPS gene; Food industries; Fundamental and applied biological sciences. Psychology; General aspects; Genetic Markers; genetically modified foods; goxv247 gene; herbicide resistance; insecticidal proteins; Methods of analysis, processing and quality control, regulation, standards; Molecular Sequence Data; nucleotide sequences; pat gene; pest resistance; Plants, Genetically Modified - genetics; plasmid vectors; Plasmids - genetics; polymerase chain reaction; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Sequence Analysis, DNA; traceability; transgenes; transgenic plants; Zea mays; Zea mays - genetics; Monocotyledones; event-specific marker; Identification; GT73 canola; PCR identification and quantification; Gramineae; Angiospermae; Bt11 maize; Transgenic plant; Quantitative analysis; Zea mays; Corn; GMO; GA21 maize; Marker; Method; Real time; Standards; Bt176 maize; Plasmid; Polymerase chain reaction; Cereal; Spermatophyta; Molecular biology; Genetically modified organism
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf0483467

Since the 18th of April 2004, two new regulations, EC/1829/2003 on genetically modified food and feed products and EC/1830/2003 on traceability and labeling of GMOs, are in force in the EU. This new, comprehensive regulatory framework emphasizes the need of an adequate tracing system. Unique identifiers, such as the transgene genome junction region or a specific rearrangement within the transgene DNA, should form the basis of such a tracing system. In this study, we describe the development of event-specific tracing systems for transgenic maize lines Bt11, Bt176, and GA21 and for canola event GT73. Molecular characterization of the transgene loci enabled us to clone an event-specific sequence into a plasmid vector, to be used as a marker, and to develop line-specific primers. Primer specificity was tested through qualitative PCRs and dissociation curve analysis in SYBR Green I real-time PCRs. The primers were then combined with event-specific TaqMan probes in quantitative real-time PCRs. Calibration curves were set up both with genomic DNA samples and the newly synthesized plasmid DNA markers. It is shown that cloned plasmid GMO target sequences are perfectly suitable as unique identifiers and quantitative calibrators. Together with an event-specific primer pair and a highly specific TaqMan probe, the plasmid markers form crucial components of a unique and straighforward tracing system for Bt11, Bt176, and GA21 maize and GT73 canola events. Keywords: GMO; event-specific marker; PCR identification and quantification; GA21 maize; Bt176 maize; Bt11 maize; GT73 canola