Zanatta, C.B.; Hoepers, A.M.; Nodari, R.O.; Agapito-Tenfen, S.Z. Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations. Foods2023, 12, 4298.
Zanatta, C.B.; Hoepers, A.M.; Nodari, R.O.; Agapito-Tenfen, S.Z. Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations. Foods 2023, 12, 4298.
Zanatta, C.B.; Hoepers, A.M.; Nodari, R.O.; Agapito-Tenfen, S.Z. Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations. Foods2023, 12, 4298.
Zanatta, C.B.; Hoepers, A.M.; Nodari, R.O.; Agapito-Tenfen, S.Z. Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations. Foods 2023, 12, 4298.
Abstract
The term new genomic techniques (NGTs), is an umbrella term to describe a variety of techniques that can alter the genetic material of an organism and that have emerged or have been developed since 2001, when the existing genetically modified organism (GMO) legislation was adopted. The analytical framework to detect GMOs in Europe is a established single harmonized procedure which is mandatory for authorization of GM food and feed, thus generating reliable, transparent and effective labelling scheme for GMO products. However, NGT products challenge the implementation and enforcement of the current regulatory system in the EU, relating in particular to the detection of NGT products that contain no foreign genetic material. Consequently, current detection methods will fail to meet minimum performance requirements. Although existing detection methods may be able to detect and quantify even small alterations in the genome, this does not necessarily confirm the distinction between products resulting from NGTs subject to the GMO legislation and other products. Therefore, this study provides a stepwise approach for the in silico prediction of PCR systems specificity by testing a bioinformatics pipeline for amplicon and primer set searches in current genomic databases. We have tested our methodology in two mutant genotypes produced by CRISPR/Cas9 in Arabidopsis thaliana. Overall, our results demonstrated that the single PCR system developed for the identification of a nucleotide insertion in the grf1-3 genotype has sufficient hits in the databases that would not allow discrimination of this mutanted event. Differently, the second mutated genotype grf8-61 which contains a -3bp deletion, found no matches in the sequence variant database. Our approach is the first step in decision making on PCR system and modification of them for empirical testings.
Keywords
genetically modified organisms; detection; site directed nucleases; traceability; CRISPR; bioinformatics
Subject
Biology and Life Sciences, Biology and Biotechnology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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