Abstract In Belgium, an official control plan was established in 2016 to detect the potential presence of an unauthorized genetically modified (GM) Bacillus subtilis RASFF2014.1249 strain in commercialized feed additive vitamin B2 products. To this end, two real-time PCR markers specific to this unauthorized genetically modified microorganism (GMM), named UGMVit-B2 and 558, were used. In the present study, the first four-year results from 67 feed additive vitamin B2 samples from the official control are presented. It includes 5 samples positive for real-time PCR methods specific to the unauthorized GM B. subtilis RASFF2014.1249 strain and has led to the RASFF2018.2755 and RASFF2019.3216 notifications. Moreover, a retrospective study using the same feed additive vitamin B2 samples was performed, allowing to provide a first picture of GM bacterial contaminations. It consisted in a first-line screening strategy gathering available PCR-based methods targeting both the B. subtilis species, frequently used to produce vitamin B2, and a set of antimicrobial resistance (AMR) genes commonly harboured as selection marker by GM bacteria used to produce microbial fermentation products. On this basis, suspicious samples contaminated with additional unknown GM bacterial strains as well as potential health and environmental risks related to the unexpected presence of full-length AMR genes could be highlighted. In addition, the possible complementary use of additional data, like chloramphenicol presence and DNA concentration, as indicators for GMM contaminations was assessed. Based on results generated in the present study, the relevance to use the proposed first-line screening strategy supplemented by indicators in order to strengthen the current control strategy was emphasized.

中文翻译：

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比利时商业化饲料添加剂维生素 B2 中含有抗微生物基因的未经授权转基因细菌的回顾性调查：挑战和解决方案

摘要 比利时于 2016 年制定了官方控制计划，以检测商业化饲料添加剂维生素 B2 产品中是否存在未经授权的转基因 (GM) 枯草芽孢杆菌 RASFF2014.1249 菌株。为此，使用了两种特定于这种未经授权的转基因微生物 (GMM) 的实时 PCR 标记，命名为 UGMVit-B2 和 558。在本研究中，展示了来自官方对照的 67 个饲料添加剂维生素 B2 样品的前四年结果。它包括 5 个针对未经授权的 GM B. subtilis RASFF2014.1249 菌株的实时 PCR 方法呈阳性的样本，并已导致 RASFF2018.2755 和 RASFF2019.3216 通知。此外，还使用相同的饲料添加剂维生素 B2 样品进行了回顾性研究，允许提供转基因细菌污染的第一张图片。它包括收集可用的基于 PCR 的方法的一线筛选策略，这些方法针对常用于生产维生素 B2 的枯草芽孢杆菌物种和一组通常被转基因细菌用作选择标记的抗菌素抗性 (AMR) 基因，用于生产微生物发酵产品。在此基础上，可以强调被其他未知转基因细菌菌株污染的可疑样本以及与全长 AMR 基因意外存在相关的潜在健康和环境风险。此外，还评估了额外数据的可能补充用途，如氯霉素的存在和 DNA 浓度，作为 GMM 污染的指标。根据本研究产生的结果，