Sciensano, Transversal activities in Applied Genomics (TAG), J. Wytsmanstraat 14, 1050 Brussels, Belgium.
Int J Food Microbiol. 2021 Jan 16;337:108913. doi: 10.1016/j.ijfoodmicro.2020.108913. Epub 2020 Oct 21.
Recently, unexpected contaminations of unauthorized genetically modified microorganisms (GMM) carrying antimicrobial resistance (AMR) genes were reported in microbial fermentation products commercialized on the food and feed chain. To guarantee the traceability and safety of the food and feed chain, whole-genome sequencing (WGS) has played a key role to prove GMM contaminations via the characterization of unnatural associations of sequences. However, WGS requires a prior microbial isolation of the GMM strain, which can be difficult to successfully achieve. Therefore, in order to avoid such bottleneck, a culture-independent approach was proposed in this study. First, the screening for the aadD gene, an AMR gene conferring a resistance to kanamycin, and for the pUB110 shuttle vector, carrying the aadD gene and commonly used to produce GMM, is performed. In case of a positive signal, DNA walking methods anchored on the two borders of the detected pUB110 shuttle vector are applied to characterize unknown flanking regions. Following to the sequencing of the generated amplicons, unnatural associations of sequences can be identified, allowing to demonstrate the presence of unauthorized GMM. The developed culture-independent strategy was successfully applied on commercialized microbial fermentation products, allowing to prove the presence of GMM contaminations in the food and feed chain.
最近，在商业化的食品和饲料链中的微生物发酵产品中，报告了携带抗微生物药物耐药性（AMR）基因的未经授权的遗传修饰微生物（GMM）的意外污染。为了保证食品和饲料链的可追溯性和安全性，全基因组测序（WGS）通过对非自然序列关联的特征描述，在证明 GMM 污染方面发挥了关键作用。然而，WGS 需要事先对 GMM 菌株进行微生物分离，这可能难以成功实现。因此，为了避免这种瓶颈，本研究提出了一种非培养方法。首先，筛选 AMR 基因 aadD 和携带 aadD 基因的 pUB110 穿梭载体，该载体用于生产 GMM。如果检测到阳性信号，则应用锚定在检测到的 pUB110 穿梭载体两端的 DNA 步行方法来表征未知的侧翼区域。对生成的扩增子进行测序后，可以识别非自然序列的关联，从而证明存在未经授权的 GMM。开发的非培养策略已成功应用于商业化的微生物发酵产品，证明了食品和饲料链中存在 GMM 污染。
