Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices

Abstract

BackgroundIn order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element.MethodsFood/feed matrices containing transgenic crops (Bt rice or MON863 maize) were analysed using the integrated DNA walking system.ResultsFirst, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples.ConclusionDue to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0191-3) contains supplementary material, which is available to authorized users.

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method

“…Among these, low copy number integration is the most favorable molecular profile for selecting the best events from putative lines (Kovalic et al, 2012). Furthermore, development of even-specific detection methods is not only useful for breeding program, but also of particular importance for bio-risk management to ensure food, feed, and environmental safety (Arne et al, 2012; Daniela et al, 2013; Fraiture et al, 2015a). …”

Current and New Approaches in GMO Detection: Challenges and Solutions

“…This approach, belonging to the PCR-based method category, has also the advantage to be fully integrated into the GMO routine analysis as the similar primers are used for the qPCR screening (detection of potential GMO presence) and the DNA walking (GMO identification). So, this simple and rapid approach could easily be applied by the enforcement laboratories, without any significant additional cost and equipment, to confirm signals previously obtained in qPCR ( Table 9 ) [ 33 , 117 , 118 ].…”

“…Since DNA walking requires less prior knowledge about the sequence of interest than conventional PCR-based methods previously described, GMO with entirely or partially known sequences could be characterized. Therefore, in targeting key elements, such as p35S and tNOS that are highly frequent in GM crops, a broad range of GMO could be characterized [ 96 , 106 , 110 , 111 , 113 , 118 , 156 ]. In order to especially identify unauthorized GMO in European Union, a DNA walking approach using primers specific to the element t35S from the pCAMBIA vector, found in approximately 30% of transgenic plants, was developed [ 33 , 117 ].…”

“…Indeed, in order to guarantee the entire representativeness of GMO present in a tested sample, all observed amplicons should be analyzed. However, the purification of the potential numerous amplicons excised from the electrophoresis gel and the subsequent Sanger sequencing could be cumbersome, especially in case of food/feed matrices containing several GMO sharing common targeted elements [ 116 , 118 , 177 ].…”

Next generation DNA sequencing based strategies; towards a new era for the traceability of endangered species and genetically modified organisms

“…Subsequently, two nested PCRs were performed for all the four reactions to gain in specificity and amount of amplified targets [254]. This approach was evaluated and validated on in-house made GM rice food mixtures and processed rice food [254,255].…”