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Most of the bacteria that are used to produce fermentation products, such as enzymes, additives and flavorings, belong to the Bacillus subtilis group. Recently, unexpected contaminations with unauthorized genetically modified (GM) bacteria (viable cells and associated DNA) that were carrying antimicrobial resistance (AMR) genes was noticed in several microbial fermentation products that have been commercialized on the food and feed market. These contaminations consisted of GM Bacillus species belonging to the B. subtilis group. In order to screen for the potential presence of such contaminations, in this study we have developed a new real-time PCR method targeting the B. subtilis group, including B. subtilis, B. licheniformis, B. amyloliquefaciens and B. velezensis. The method’s performance was successfully assessed as specific and sensitive, complying with the Minimum Performance Requirements for Analytical Methods of GMO Testing that is used as a standard by the GMO enforcement laboratories. The method’s applicability was also tested on 25 commercial microbial fermentation products. In addition, this method was developed to be compatible with the PCR-based strategy that was recently developed for the detection of unauthorized GM bacteria. This taxon-specific method allows the strengthening of the set of screening markers that are targeting key sequences that are frequently found in GM bacteria (AMR genes and shuttle vector), reinforcing control over the food and feed chain in order to guarantee its safety and traceability.

Wastewater-based surveillance can be used as a complementary method to other SARS-CoV-2 surveillance systems. It allows the emergence and spread of infections and SARS-CoV-2 variants to be monitored in time and place. This study presents an RT-ddPCR method that targets the T19I amino acid mutation in the spike protein of the SARS-CoV-2 genomes, which is specific to the BA.2 variant (omicron). The T19I assay was evaluated both in silico and in vitro for its inclusivity, sensitivity, and specificity. Moreover, wastewater samples were used as a proof of concept to monitor and quantify the emergence of the BA.2 variant from January until May 2022 in the Brussels-Capital Region which covers a population of more than 1.2 million inhabitants. The in silico analysis showed that more than 99% of the BA.2 genomes could be characterized using the T19I assay. Subsequently, the sensitivity and specificity of the T19I assay were successfully experimentally evaluated. Thanks to our specific method design, the positive signal from the mutant probe and wild-type probe of the T19I assay was measured and the proportion of genomes with the T19I mutation, characteristic of the BA.2 mutant, compared to the entire SARS-CoV-2 population was calculated. The applicability of the proposed RT-ddPCR method was evaluated to monitor and quantify the emergence of the BA.2 variant over time. To validate this assay as a proof of concept, the measurement of the proportion of a specific circulating variant with genomes containing the T19I mutation in comparison to the total viral population was carried out in wastewater samples from wastewater treatment plants in the Brussels-Capital Region in the winter and spring of 2022. This emergence and proportional increase in BA.2 genomes correspond to what was observed in the surveillance using respiratory samples; however, the emergence was observed slightly earlier, which suggests that wastewater sampling could be an early warning system and could be an interesting alternative to extensive human testing.

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