Еvolution in the GMO detection and quantificati

Abstract

Since the nineties, genetic modification became part of the food and feed production process. In that period, when the GM crops were for the first time introduced into the environment, the emphasis was placed mainly on the risk assessment. High importance was also given to the tracкability and monitoring of the GMOs. The main principle which was established in GMO detection and control was case-by case and crop-by-crop approach. The GMO detection can be performed at a protein and nucleic acid level. The protein analysis was suitable more for screening of non or low-processed food and feed products. These techniques are based on detecting proteins produced by the inserted genes. In general, there are two different protein techniques: lateral flow strip tests as only qualitative and the Enzyme-Linked Immunosorbent Assay (ELISA) as semi-quantitative. Both are event specific and antibody-based methods used for measuring the presence of the GM protein in the unprocessed material such as seed, grain, or leaves. Strip tests use a detection surface comprised of immobilized GM protein-specific antibodies on a solid strip and it is suitable for field testing with low sensitivity (Limit of detection 0.1 % – 1.0 %). The ELISA test uses a detection surface comprised of immobilized GM protein-specific antibodies in a multi-well solid plate format with higher sensitivity compared to strip test (Limit of detection 0.01 % – 0.1 %). Our results, where as a target molecule served CP4 EPSPS enzyme showed that strip test detected the GM presence of 0.6 m/m %, while the ELISA method revealed 0.1 m/m % of GMO, but with a certain limitations for an accurate quantification. The GMO analysis on a nucleic acid level is based on the detection of a part of the inserted DNA and it can be done using promoters and terminators, or targeted DNA sequence of the inserted gene(s). Both approaches are PCR based and they could be qualitative when using the conventional PCR or quantitative when using the real-time PCR instruments (qPCR). Regarding the first one we developed a duplex PCR screening for the GM presence in one single step, instead of performing two different assays (one for the endogen and one for the transgene) both steps were performed at once in a single tube. In such a way we reduced the costs with an elimination of all negative samples from the further qPCR analysis. Currently, we are using 4 plex RT PCR using two promoters, one terminator and internal amplification control where “the results of quantitative analysis should be expressed as the number of target DNA sequences per target taxon specific sequences. It is calculated in terms of haploid genomes” according to EC Regulation 1830/2003 which is the crucial document for harmonization of GMO detection and quantification. Nowadays, clear guidelines for method acceptance and performance are developed by European Union Reference Laboratory for GM Food and Feed using appropriate Certified Reference Materials (CRMs) produced by JRC - Geel. The new achievements in GMO quantification gained using the so called digital PCR increased the level of sensitivity of those techniques. Indeed, many difficulties in the GMO detection are generated by the stacked genes and the process will become even more delicate after wider application of the CRISPR technology.