Strategy to Detect Genetically Modified Bacteria Carrying Tetracycline Resistance Gene in Fermentation Products
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Strategy to Detect Genetically Modified Bacteria Carrying Tetracycline Resistance Gene in Fermentation Products Marie-Alice Fraiture 1 & Marie Deckers 1 & Nina Papazova 1 & Nancy H. C. Roosens 1 Received: 2 March 2020 / Accepted: 14 June 2020 # The Author(s) 2020
Abstract Unexpected contaminations of unauthorized genetically modified microorganisms (GMM) harbouring antimicrobial resistance (AMR) genes in food and feed enzymes, additives and flavourings commercialized on the European market have recently alerted the competent authorities regarding the food and feed safety. At the control level, we have therefore proposed a PCR-based strategy as first line screening targeting GMM carrying AMR genes in order to help enforcement laboratories. The potential presence of frequently used AMR genes is first investigated, using real-time PCR. In case of a suspicious matrix, the full-length of the detected AMR genes is then determined, using conventional PCR followed by Sanger sequencing, allowing to support the competent authorities in their evaluation related to potential health risks. In this study, PCR methods targeting an additional key AMR gene, being the tet-L gene (GenBank: D00946.1) conferring a resistance to tetracycline, were developed and successfully assessed in terms of specificity, sensitivity and applicability. In integrating these PCR methods, the proposed PCR-based strategy, initially targeting two key AMR genes conferring a resistance to chloramphenicol (GenBank: NC_002013.1) and kanamycin (GenBank: M19465.1), is consequently strengthened, allowing the coverage of a larger spectrum of potential GMM contaminations in microbial fermentation products. Keywords Tetracycline resistance gene . Food and feed enzymes . Additives and flavourings . Genetically modified microorganisms . PCR-based strategy . Full-length AMR gene . Food and feed safety
Introduction Recently, the unauthorized presence of living strains or associated recombinant DNA from genetically modified microorganisms (GMM) have been discovered in microbial fermentation products commercialized on the European (EU) food and feed market (RASFF portal). For instance, in 2014, 2018 Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12161-020-01803-6) contains supplementary material, which is available to authorized users. * Nancy H. C. Roosens [email protected] Marie-Alice Fraiture [email protected] Marie Deckers [email protected] Nina Papazova [email protected] 1
Sciensano, Transversal Activities in Applied Genomics (TAG), J. Wytsmanstraat 14, 1050 Brussels, Belgium
and 2019, feed additives composed of vitamin B2 were contaminated by a genetically modified (GM) vitamin B2– producing Bacillus subtilis (RASFF2014.1249, RASFF2014.1360, RASFF2014.1657, RASFF2018.2755, RASFF2019.0793 and RASFF2019.3216). In addition, in 2019, a food enzyme preparation containing a protease was contaminated by a GM protease-producing Bacillus velezensis strain (RASFF2019.3332). These no
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