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，在食品和饲料链上商业化的微生物发酵产品中，报告了带有抗菌素抗性（AMR）基因的未经授权的转基因微生物（GMM）的意外污染。为了确保食品和饲料链的可追溯性和安全性，全基因组测序（WGS）发挥了关键作用，通过对序列的非自然关联进行表征来证明GMM污染。但是，WGS需要事先对GMM菌株进行微生物分离，这可能很难成功实现。因此，为了避免这种瓶颈，本研究提出了一种与文化无关的方法。首先，筛选aadD基因，赋予卡那霉素抗性的AMR基因和pUB110穿梭载体，进行了通常用于产生GMM的aadD基因。在阳性信号的情况下，将锚定在检测到的pUB110穿梭载体两个边界上的DNA步行方法用于表征未知的侧翼区域。在对所产生的扩增子进行测序之后，可以鉴定出不自然的序列关联，从而证明存在未经授权的GMM。所开发的与培养无关的策略已成功应用于商业化的微生物发酵产品，从而证明了食品和饲料链中存在GMM污染。