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

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, from G to T, in the AHAS1C gene which confers herbicide tolerance to those plants. 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 the EU regulations. We thoroughly assessed the PCR method on the basis of 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 OSR line 40K. 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 spiked at working amount levels (100 to 300 ng per reaction). Significant non-specific PCR amplifications with non-intended PCR products as template DNA and numerous OSR materials including genomic DNA from wild types, Clearfield OSR varieties and genetically modified (GM) lines as well as from wild radish (Raphanus raphanistrum) were observed. Non-specific amplifications were detected by three ISO/IEC 17025 accredited reference laboratories in tests using different master mixes and real-time PCR cycler models. The assessment shows that the method does not meet the minimum performance requirements for qualitative PCR methods used for GMO testing. Consequently, the method is not fit-for-purpose for use in official control of genetically modified food, feed and seeds. Suggestions are provided for specific validation conditions under which methods for detecting genome-edited organisms should be validated.

WEIDNER Christopher;  SOPHIA Edelmann;  MOORE D.;  LIESKE Kathrin;  SAVINI Cristian;  JACCHIA Sara;  SACCO Maria-Grazia;  MAZZARA Marco;  LAEMKE Joern;  ECKERMANN Kolja Neil;  EMONS Hendrik;  MANKERTZ Joachim;  GROHMANN Lutz; 

2022-12-12

SPRINGER

JRC126296

1936-9751 (online),   

https://link.springer.com/article/10.1007/s12161-022-02237-y#citeas,    https://publications.jrc.ec.europa.eu/repository/handle/JRC126296,   

10.1007/s12161-022-02237-y (online),