Assessment of the Real-Time PCR Method Claiming to be Specific for Detection and Quantification of the First Commercialised Genome-Edited Plant

Abstract

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.

Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations

“…As expected, the BLAST analysis of amplicon grf1-3 indicated that the single nucleotide polymorphism was the only difference in 35 sequence hits with 99.8% similarity. Similar results were found by [13] when analyzing amplicons of primer sets for the detection of gene-edited canola. In their study, the BLAST search produced hits in B. oleracea and B. carinata, where the only difference between the targets was the last nucleotide at position 22.…”

“…The technical complexity of designing a PCR system capable of distinguishing specific sequences with accuracy and reliability requires both in silico and experimental testing. Some studies have successfully distinguished NGT canola and rice varieties using PCR [11,13,14].…”

Genome editing around the globe: An update on policies and perceptions

“…Several of those already have a GEd regulation in place which allows exemptions of some GEd products from strict GMO regulations including labeling. This situation is contrary to the legislation in Europe, and the situation may worsen, as clear detection and identification of such products in goods is currently not possible without technical details (unique analytical reference data), and will not be possible in the near future ( Grohmann et al, 2019 ; Weidner et al, 2022 ). In the past, many countries remained on hold waiting for Europe to make a move toward a clear regulation (see Sprink et al, 2016 ).…”

The Role of Italy in the Use of Advanced Plant Genomic Techniques on Fruit Trees: State of the Art and Future Perspectives

“…However, while most of the extra-European countries have adopted or are adopting legislations that differentiate many NGT products from GMOs, making their authorization for the market more streamlined and faster, the current European regulatory framework would constitute a strong obstacle to the development of NGTs in Europe [ 214 ]. On a more technical level, the EC identified substantial issues concerning traceability of some products obtained through NGTs because it is possible to analytically detect single-point mutations in a target sequence but not their origin, whether natural or induced in the laboratory [ 215 ]. Therefore, to keep pace with the scientific and technological development and favor European economic and social progress, the EC has decided to test whether the conditions exist in Europe for a change in the legislative framework on NGTs in plants.…”