Event Specific Transgene Detection in Bt11 Corn by Quantitative PCR at the Integration Site

The genetically modified corn Bt11 from Novartis is widely cultivated for production of foodstuffs or fodder. Due to the insertion and expression of a 6.3 kb sequence containing a Bacillus thuringiensis (Bt)-transgene coding for a synthetic cryIA(b) -endotoxin, the Bt11-corn is resistant against lepidopteran insects, especially against the European corn borer. As labelling of genetically modified foodstuffs is mandatory in many European countries, several methods to detect genetically modified organisms (GMOs) qualitatively have been described. Since these `conventionalʹ detection systems are all based on the polymerase chain reaction (PCR) amplifying unique sequences within the integrated transgenic constructs, they may lead to ambiguous results in qualitative and especially in quantitative analysis of GMOs in foodstuffs and fodder. In this study, we report the characterization of the genomic sequence at the 5ʹ-site of the integrated transgenic sequence in the Bt11-corn genome using inverse PCR. The integration border was subsequently used to develop a novel and unambiguous PCR detection system covering the integration border at the 5ʹ site of the transgene. The genomic sequence showed high similarities with a corn-specific 180 bp knob-associated repeat region. By using one primer annealing exactly at the integration border between the transgenic construct and the corn genome and the other within the vector sequence of the transgenic construct, a 207 bp product was amplified. After insertion of a 22 bp fragment, the amplification product was subsequently used as a competitor in quantitative competitive PCR (qcPCR). Finally, the qcPCR-system was calibrated to an equivalence point of 1% Bt11-DNA.