Abstract
The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). In parallel, the number of reports of unauthorised GMOs (UGMOs) is gradually increasing. There is a lack of specific detection methods for UGMOs, due to the absence of detailed sequence information and reference materials. In this research, an adapted genome walking approach was developed, called ALF: Amplification of Linearly-enriched Fragments. Coupling of ALF to NGS aims for simultaneous detection and identification of all GMOs, including UGMOs, in one sample, in a single analysis. The ALF approach was assessed on a mixture made of DNA extracts from four reference materials, in an uneven distribution, mimicking a real life situation. The complete insert and genomic flanking regions were known for three of the included GMO events, while for MON15985 only partial sequence information was available. Combined with a known organisation of elements, this GMO served as a model for a UGMO. We successfully identified sequences matching with this organisation of elements serving as proof of principle for ALF as new UGMO detection strategy. Additionally, this study provides a first outline of an automated, web-based analysis pipeline for identification of UGMOs containing known GM elements.
Highlights
The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs)
A protocol was developed for the identification of unknown GMO-related sequences starting from known GMO elements, called ALF: Amplification of Linearly-enriched Fragments (Fig. 1)
To increase the reliability of the quantification outcomes, Joint Research Centre (JRC)-validated qPCR methods were used in combination with certified reference material for >99.05% MON88107. qPCRs were performed before the procedure in the starting material (SM), after Linear enrichment (LE), and after snPCR
Summary
The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). Additional analyses are necessary to determine whether the identified GMO element is linked to an unknown, unauthorised GMO, or rather to, for instance, the native organism of the element In these cases, additional experiments will be required, that are currently usually based on variants of gene walking (GW) strategies where the unexplained elements can be used as a starting point for GW to obtain adjacent sequence information. This sequence information could lead to the identification of a specific UGMO, especially when the sequence information stretches into the flanking genomic region of the GMO insert. Very recently this method was succesfully applied in combination with NGS and more complex GMO mixtures[21]
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