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Strategy to Detect Genetically Modified Bacteria Carrying Tetracycline Resistance Gene in Fermentation Products
Food Analytical Methods ( IF 2.9 ) Pub Date : 2020-07-07 , DOI: 10.1007/s12161-020-01803-6
Marie-Alice Fraiture , Marie Deckers , Nina Papazova , Nancy H. C. Roosens

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.



中文翻译:

检测发酵产物中携带四环素抗性基因的转基因细菌的策略

在欧洲市场上商业化的食品和饲料酶,添加剂和调味剂中,带有抗菌抗性(AMR)基因的未经授权的转基因微生物(GMM)受到意料之外的污染,最近已就食品和饲料安全向主管部门发出警告。因此,在对照水平上,我们提出了一种基于PCR的策略,作为针对携带AMR基因的GMM的一线筛选,以帮助执法实验室。首先使用实时PCR研究经常使用的AMR基因的潜在存在。在可疑基质的情况下,然后使用常规PCR和Sanger测序方法确定检测到的AMR基因的全长,从而可以支持主管当局进行有关潜在健康风险的评估。在这个研究中,已开发出赋予四环素抗性的tet-L基因(GenBank:D00946.1),并已在特异性,敏感性和适用性方面进行了成功评估。通过整合这些PCR方法,基于PCR的策略最初针对了两个关键的AMR基因,从而赋予了对氯霉素(GenBank:NC_002013.1)和卡那霉素(GenBank:M19465.1)的抗性,从而覆盖了微生物发酵产品中更大范围的潜在GMM污染。

更新日期:2020-07-07
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