Christopher Weidner

The Working Group on Method Performance Requirements (WG-MPR) of the European Network of GMO Laboratories (ENGL) and the EU Reference Laboratory for Genetically Modified Food and Feed (EURL GMFF) have extended the scope of its guidance document on the “Definition of minimum performance requirements for analytical methods of GMO testing”. This document establishes criteria for the development and validation of methods for GMO analysis, both for EU market authorization applications and for official food and feed control. The guidance outlines definitions and performance requirements for methods used to detect and quantify GMOs and has recently been expanded to include digital PCR methods, the detection of genetically modified animals, and the analysis of products developed using new mutagenesis techniques. The guidance document is freely available on the EURL GMFF website (https://publications.jrc.ec.europa.eu/repository/handle/JRC125975 accessed 25.02.2025).

Under current EU legislation, genetically modified organisms (GMOs) and derived food and feed products must be authorized as GM food, feed, or seed and appropriate detection methods must be made available for use in official controls. A Real-Time PCR method has recently been published by Chhalliyil et al. claiming to be specific for the detection and identification of genome-edited oilseed rape (OSR) lines commercialized in North America. In a previous study, we have independently assessed this method in three reference laboratories for sensitivity, specificity, and robustness. We found that the method does not meet all the minimum performance requirements (MPR) for GMO testing in the EU, which contradicts the claims of the method developer. Here we show, in addition to the previously published method assessment study that a modified DNA extraction is not the reason for the contradictory findings and does not affect the specificity of the method. We also discuss the procedures recently proposed by the method developers for interpreting PCR results with high Cq values.

The international working group “Development of methods for identification of foodstuffs produced by means of genetic engineering techniques” was established pursuant to § 64 of the German Food and Feed Code (LFGB). It has developed a guideline for implementing and verifying digital PCR methods previously validated as real-time PCR techniques. While event-specific methods in testing of genetically modified organisms (GMO) have traditionally relied on real-time PCR, new digital PCR technologies offer significant advantages and therefore, leading to an increased use by control laboratories. This guideline provides practical recommendations for transferring the real-time PCR to digital PCR and for verifying the digital PCR method. The guideline is applicable to analysis of GMO in food, feed and seed and is freely available on the website of the Federal Office of Consumer Protection and Food Safety (BVL).

The diversity of genetically modified plants used for food and feed is increasing worldwide. For the detection and control of these products, efficient and reliable analytical tools are a prerequisite. This can be done by screening for specific DNA-elements and constructs characteristic of transgenic plants. In the past, numerous methods have already been published. However, several genetically modified plants are not covered by common screening methods. Here, a new qualitative triplex real-time polymerase chain reaction (PCR) method is presented, detecting two transgene flanking sequences and the transition between the Cassava Vein Mosaic Virus Promotor (P-CsVMV) and the phosphinothricin-N-acetyltransferase (pat) gene. These sequences are present in several transgenic plants and therefore, the described triplex method can be used as a screening tool to guide further analysis and increase the efficiency of the analysis strategy for GMO detection. The method is characterized by high specificity, sensitivity and robustness and is provided as a ring-trial validated method in the Official Collection of Methods according to the German Food and Feed Act.

For the impact of genomic testing from liquid biopsies to be maximized, mechanisms to ensure reproducible and comparable test performance will be required. This can be established and maintained through reference measurement procedures and materials with property values that are internationally comparable through traceability to a common standard. To achieve this objective, an interlaboratory study was organised to explore digital PCR (dPCR) for standardisation of cell-free DNA (cfDNA) quantification. Blinded samples of wild-type/variant mixtures of two DNA sequences ( BRAF p.V600E single nucleotide variant or EGFR exon 19 deletion) were provided to 12 laboratories. Laboratories independently designed and applied dPCR assays to determine absolute and relative quantities, with no guidance provided to harmonise the approach. The mean and coefficient of variation (CV) of copy number concentrations for variant sequences were 18 copies/μL (CV 7.2%) ( BRAF variant sample) and 9 copies/μL (CV 25%) ( EGFR variant sample) while the mean variant allele frequencies (vAF) were 8.0% (CV 5.3%) and 0.080% (CV 29%) respectively. This study demonstrated that dPCR was capable of exceptional technical accuracy for variant copy number concentration and vAF, even when different assays and platforms were used. This implies that dPCR offers a unique analytical methodology that can be deployed globally in supporting comparability for cfDNA testing based on the existing framework of the International System of units of measurement.

For the in-house validation of a droplet digital PCR method, a factorial experimental design was implemented. This design serves different purposes. On the one hand, it is an efficient design in relation to the workload involved in achieving a desirable level of reliability of variance estimates. On the other hand, it allows a partitioning of total variance into different components, thus providing information regarding the dominant sources of random variation. The statistical modelling reflects the actual measurement mechanism, establishing relationships between nominal target DNA copies per well, the range of variation of copy numbers per droplet, probability of detection values, and estimated numbers of copies.

A real-time PCR method was recently published with a claim to be specific for the detection and identification of some genome-edited oilseed rape (OSR) lines commercialised in North America. The method was designed to detect a single base mutation in the AHAS1C gene, which confers herbicide tolerance. The authors claim that the method is event-specific for the genome-edited OSR line 5715 and fulfils all requirements for GMO analytical methods according to EU regulations. We have thoroughly assessed the method in relation to the minimum performance requirements (MPR) established by the European Network of GMO Laboratories (ENGL). The method was found to be sufficiently sensitive and robust when tested with pure genomic DNA of the OSR line 40 K. However, our results show that the method is not event-specific and detects also OSR lines carrying the same point mutation caused by somaclonal variation. Moreover, impaired robustness was observed using non-modified genomic DNA at the amount specified in the original protocol. Significant non-specific PCR amplifications with PCR products as non-target template DNA and with genomic DNA from numerous OSR varieties as well as from wild radish were found by three ISO/IEC 17025 accredited reference laboratories in tests using different master mixes and PCR cycler models. The assessment shows that the method does not meet the MPR for qualitative PCR methods and therefore is not fit-for-purpose for official controls of genetically modified products in the EU. Suggestions are provided for conditions under which analytical methods for genome-edited organisms should be validated.