Detection by real-time PCR and pyrosequencing of thecry1Ab andcry1Ac genes introduced in genetically modified (GM) constructs

The presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random

Detection and identification of transgenic events by next generation sequencing combined with enrichment technologies.

Next generation sequencing (NGS) is a promising tool for analysing the quality and safety of food and feed products. The detection and identification of genetically modified organisms (GMOs) is complex, as the diversity of transgenic events and types of structural elements introduced in plants continue to increase. In this paper, we show how a strategy that combines enrichment technologies with NGS can be used to detect a large panel of structural elements and partially or completely reconstruct the new sequence inserted into the plant genome in a single analysis, even at low GMO percentages. The strategy of enriching sequences of interest makes the approach applicable even to mixed products, which was not possible before due to insufficient coverage of the different genomes present. This approach is also the first step towards a more complete characterisation of agrifood products in a single analysis