Development of a reverse transcriptase digital droplet polymerase chain reaction-based approach for SARS-CoV-2 variant surveillance in wastewater.

An urgent need for effective surveillance strategies arose due to the global emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although vaccines and antivirals are available, concerns persist about the evolution of new variants with potentially increased infectivity, transmissibility, and immune evasion. Therefore, variant monitoring is crucial for public health decision-making. Wastewater-based surveillance has proven to be an effective tool to monitor SARS-CoV-2 variants within populations. Specific SARS-CoV-2 variants are detected and quantified in wastewater in this study using a reverse transcriptase digital droplet polymerase chain reaction (RT-ddPCR) approach. The 11 designed assays were first validated in silico using a substantial dataset of high-quality SARS-CoV-2 genomes to ensure comprehensive variant coverage. The assessment of the sensitivity and specificity with reference material showed the capability of the developed assays to reliably identify target mutations while minimizing false positives and false negatives. The applicability of the assays was evaluated using wastewater samples from a wastewater treatment plant in Ghent, Belgium. The quantification of the specific mutations linked to the variants of concern present in these samples was calculated using these assays based on the detection of single mutations, which confirms their use for real-world variant surveillance. In conclusion, this study provides an adaptable protocol to monitor SARS-CoV-2 variants in wastewater with high sensitivity and specificity. Its potential for broader application in other viral surveillance contexts highlights its added value for rapid response to emerging infectious diseases. PRACTITIONER POINTS: Robust RT-ddPCR methodology for specific SARS-CoV-2 variants of concern detection in wastewater. Rigorous validation that demonstrates high sensitivity and specificity. Demonstration of real-world applicability using wastewater samples. Valuable tool for rapid response to emerging infectious diseases.

Pilot market surveillance of GMM contaminations in alpha-amylase food enzyme products: A detection strategy strengthened by a newly developed qPCR method targeting a GM Bacillus licheniformis producing alpha-amylase.

Using high-throughput metagenomics on commercial microbial fermentation products, DNA from a new unauthorized genetically modified microorganism (GMM), namely the GM B. licheniformis strain producing alpha-amylase (GMM alpha-amylase2), was recently discovered and characterized. On this basis, a new qPCR method targeting an unnatural association of sequences specific to the GMM alpha-amylase2 strain was designed and developed in this study, allowing to strengthen the current GMM detection strategy. The performance of the newly developed qPCR method was assessed for its specificity and sensitivity to comply with the minimum performance requirements established by the European Network of GMO Laboratories for GMO analysis. Moreover, the transferability of the in house validated qPCR method was demonstrated. Finally, its applicability was confirmed by a pilot market surveillance of GMM contaminations conducted for the first time on 40 alpha-amylase food enzyme products labelled as containing alpha-amylase. This pilot market surveillance allowed also to highlight numerous contaminations with GMM alpha-amylase2, including frequent cross-contaminations with other GMM strains previously characterized. In addition, the presence of full-length AMR genes, raising health concerns, was also reported.

Retrospective surveillance of viable Bacillus cereus group contaminations in commercial food and feed vitamin B2 products sold on the Belgian market using whole-genome sequencing.

Bacillus cereus is a spore-forming bacterium that occurs as a contaminant in food and feed, occasionally resulting in food poisoning through the production of various toxins. In this study, we retrospectively characterized viable B. cereus sensu lato (s.l.) isolates originating from commercial vitamin B2 feed and food additives collected between 2016 and 2022 by the Belgian Federal Agency for the Safety of the Food Chain from products sold on the Belgian market. In total, 75 collected product samples were cultured on a general medium and, in case of bacterial growth, two isolates per product sample were collected and characterized using whole-genome sequencing (WGS) and subsequently characterized in terms of sequence type (ST), virulence gene profile, antimicrobial resistance (AMR) gene profile, plasmid content, and phylogenomic relationships. Viable B. cereus was identified in 18 of the 75 (24%) tested products, resulting in 36 WGS datasets, which were classified into eleven different STs, with ST165 (n = 10) and ST32 (n = 8) being the most common. All isolates carried multiple genes encoding virulence factors, including cytotoxin K-2 (52.78%) and cereulide (22.22%). Most isolates were predicted to be resistant to beta-lactam antibiotics (100%) and fosfomycin (88.89%), and a subset was predicted to be resistant to streptothricin (30.56%). Phylogenomic analysis revealed that some isolates obtained from different products were closely related or even identical indicating a likely common origin, whereas for some products the two isolates obtained did not show any close relationship to each other or other isolates found in other products. This study reveals that potentially pathogenic and drug-resistant B. cereus s.l. can be present in food and feed vitamin B2 additives that are commercially available, and that more research is warranted to assess whether their presence in these types of products poses a threat to consumers.

Targeted High-Throughput Sequencing Enables the Detection of Single Nucleotide Variations in CRISPR/Cas9 Gene-Edited Organisms.

Similar to genetically modified organisms (GMOs) produced by classical genetic engineering, gene-edited (GE) organisms and their derived food/feed products commercialized on the European Union market fall within the scope of European Union Directive 2001/18/EC. Consequently, their control in the food/feed chain by GMO enforcement laboratories is required by the competent authorities to guarantee food/feed safety and traceability (2003/1829/EC; 2003/1830/EC). However, their detection is potentially challenging at both the analytical and interpretation levels since this requires methodological approaches that can target and detect a specific single nucleotide variation (SNV) introduced into a GE organism. In this study, we propose a targeted high-throughput sequencing approach, including (i) a prior PCR-based enrichment step to amplify regions of interest, (ii) a sequencing step, and (iii) a data analysis methodology to identify SNVs of interest. To investigate if the performance of this targeted high-throughput sequencing approach is compatible with the performance criteria used in the GMO detection field, several samples containing different percentages of a GE rice line carrying a single adenosine insertion in OsMADS26 were prepared and analyzed. The SNV of interest in samples containing the GE rice line could successfully be detected, both at high and low percentages. No impact related to food processing or to the presence of other crop species was observed. The present proof-of-concept study has allowed us to deliver the first experimental-based evidence indicating that the proposed targeted high-throughput sequencing approach may constitute, in the future, a specific and sensitive tool to support the safety and traceability of the food/feed chain regarding GE plants carrying SNVs.

Metagenomic Characterization of Multiple Genetically Modified Bacillus Contaminations in Commercial Microbial Fermentation Products.

Genetically modified microorganisms (GMM) are frequently employed for manufacturing microbial fermentation products such as food enzymes or vitamins. Although the fermentation product is required to be pure, GMM contaminations have repeatedly been reported in numerous commercial microbial fermentation produce types, leading to several rapid alerts at the European level. The aim of this study was to investigate the added value of shotgun metagenomic high-throughput sequencing to confirm and extend the results of classical analysis methods for the genomic characterization of unauthorized GMM. By combining short- and long-read metagenomic sequencing, two transgenic constructs were characterized, with insertions of alpha-amylase genes originating from B. amyloliquefaciens and B. licheniformis, respectively, and a transgenic construct with a protease gene insertion originating from B. velezensis, which were all present in all four investigated samples. Additionally, the samples were contaminated with up to three unculturable Bacillus strains, carrying genetic modifications that may hamper their ability to sporulate. Moreover, several samples contained viable Bacillus strains. Altogether these contaminations constitute a considerable load of antimicrobial resistance genes, that may represent a potential public health risk. In conclusion, our study showcases the added value of metagenomics to investigate the quality and safety of complex commercial microbial fermentation products.

Detection and identification of authorized and unauthorized GMOs using high-throughput sequencing with the support of a sequence-based GMO database.

The increasing number and diversity of genetically modified organisms (GMOs) for the food and feed market calls for the development of advanced methods for their detection and identification. This issue can be addressed by next generation sequencing (NGS). However, the efficiency of NGS-based strategies depends on the availability of bioinformatic methods to find sequences of the transgenic insert and junction regions, which is a challenging topic. To facilitate this task, we have developed Nexplorer, a sequence-based database in which annotated sequences of GM events are stored in a structured, searchable and extractable format. As a proof of concept, we have developed a methodology for the analysis of sequencing data of DNA walking libraries of samples containing GMOs using the database. The efficiency of the method has been tested on datasets representing various scenarios that can be encountered in routine GMO analysis. Database-guided analysis allowed obtaining detailed and reliable information with limited hands-on time. As the database allows for efficient analysis of NGS data, it paves the way for the use of NGS sequencing technology to aid routine detection and identification of GMO.

Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution.

The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.

Characterization of Genetically Modified Microorganisms Using Short- and Long-Read Whole-Genome Sequencing Reveals Contaminations of Related Origin in Multiple Commercial Food Enzyme Products.

Despite their presence being unauthorized on the European market, contaminations with genetically modified (GM) microorganisms have repeatedly been reported in diverse commercial microbial fermentation produce types. Several of these contaminations are related to a GM Bacillus velezensis used to synthesize a food enzyme protease, for which genomic characterization remains currently incomplete, and it is unknown whether these contaminations have a common origin. In this study, GM B. velezensis isolates from multiple food enzyme products were characterized by short- and long-read whole-genome sequencing (WGS), demonstrating that they harbor a free recombinant pUB110-derived plasmid carrying antimicrobial resistance genes. Additionally, single-nucleotide polymorphism (SNP) and whole-genome based comparative analyses showed that the isolates likely originate from the same parental GM strain. This study highlights the added value of a hybrid WGS approach for accurate genomic characterization of GMM (e.g., genomic location of the transgenic construct), and of SNP-based phylogenomic analysis for source-tracking of GMM.

Food Enzyme Database (FEDA): a web application gathering information about food enzyme preparations available on the European market.

Following the European Commission No. 1332/2008 regulation and the consequent necessity of a scientific evaluation of food enzymes (FEs) for their approval for sale on the European Union market, many FE dossiers have been submitted to the European Commission and various documents currently co-exist. In order to centralize all relevant information in one structured location that is easily accessible to support enforcement laboratories and the competent authorities, we developed a web application, called Food Enzyme Database (FEDA). FEDA allows searching and collection of information originating from many different sources in one centralized portal. Queries can be performed using key information types, which include information on the producing company, production source (strain type, genetically modified microorganism status), type of enzyme protein and evaluation status with employed evaluation criteria. The database contains all current publicly available information. Centralizing all information coupled with intuitive searching functionality also allows the generation of general statistics regarding the current market situation. FEDA is open access and is freely available at the following location: https://feda.sciensano.be. Database URL : https://feda.sciensano.be.

Development of a real-time PCR marker targeting a new unauthorized genetically modified microorganism producing protease identified by DNA walking.

A PCR-based DNA walking analysis was performed on a protease product suspected to contain a new unauthorized genetically modified microorganism (GMM). Though the characterization of unnatural associations of sequences between the pUB110 shuttle vector and a Bacillus amyloliquefaciens gene coding for a protease, the presence of the GMM was shown. Based on these sequences of interest, a real-time PCR marker was developed to target specifically the newly discovered GMM, namely GMM protease2. The performance of the real-time PCR marker was assessed in terms of specificity and sensitivity. The applicability of the real-time PCR GMM protease2 marker was also demonstrated on microbial fermentation products. To confirm its use by other GMO enforcement laboratories, the transferability of the in-house validated real-time PCR marker was demonstrated by assays performed by an external laboratory.

Non-Invasive versus Invasive Samples for Zika Virus Surveillance: A Comparative Study in New Caledonia and French Guiana in 2015-2016.

Zika virus, an arbovirus responsible for major outbreaks, can cause serious health issues, such as neurological diseases. In the present study, different types of samples (serum, saliva, and urine), collected in 2015-2016 in New Caledonia and French Guiana from 53 patients presenting symptoms and clinical signs triggered by arbovirus infections, were analyzed using a recently developed, and in-house validated, 4-plex RT-qPCR TaqMan method for simultaneous detection and discrimination of the Zika and Chikungunya viruses. Subsequently, statistical analyses were performed in order to potentially establish recommendations regarding the choice of samples type to use for an efficient and early stage Zika infection diagnosis. On this basis, the use of only urine samples presented the highest probability to detect viral RNA from Zika virus. Moreover, such a probability was improved using both urine and saliva samples. Consequently, the added value of non-invasive samples, associated with a higher acceptance level for collection among patients, instead of serum samples, for the detection of Zika infections was illustrated.

A shotgun metagenomics approach to detect and characterize unauthorized genetically modified microorganisms in microbial fermentation products.

The presence of a genetically modified microorganism (GMM) or its DNA, often harboring antimicrobial resistance (AMR) genes, in microbial fermentation products on the market is prohibited by European regulations. GMMs are currently screened for through qPCR assays targeting AMR genes and vectors, and then confirmed by targeting known specific GM constructs/events. However, when the GMM was not previously characterized and an isolate cannot be obtained, its presence cannot be proven. We present a metagenomics approach capable of delivering the proof of presence of a GMM in a microbial fermentation product, with characterization based on the detection of AMR genes and vectors, species and unnatural associations in the GMM genome. In our proof-of-concept study, this approach was performed on a case with a previously isolated and sequenced GMM, an unresolved case for which no isolate was obtained, and a non-GMM-contaminated sample, all representative for the possible scenarios to occur in routine setting. Both short and long read sequencing were used. This workflow paves the way for a strategy to detect and characterize unknown GMMs by enforcement laboratories.

DNA walking strategy to identify unauthorized genetically modified bacteria in microbial fermentation products.

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.

Are antimicrobial resistance genes key targets to detect genetically modified microorganisms in fermentation products?

As genetically modified microorganisms (GMM), commonly used by the food and feed industry to produce additives, enzymes and flavourings, are frequently harbouring antimicrobial resistance (AMR) genes as selection markers, health and environmental concerns were consequently raised. For this reason, the interest of the competent authorities to control such microbial fermentation products has strongly increased, especially since several recent accidental contaminations of unauthorized GMM, or associated recombinant DNA, in bacterial fermentation products intended for the European food and feed chain. However, no global screening strategy is currently available in enforcement laboratories to assess the presence of GMM harbouring AMR genes and/or the presence of full-length AMR genes. Moreover, the confidentiality of the related GMM dossiers strongly hampers the development of methods to perform such control. To overcome this issue, an analysis of related publicly available patents was performed in this study to identify all reported AMR genes. On this basis, the aminoglycoside adenyltransferase (aadD) gene, conferring a resistance to both kanamycin and neomycin, was identified as a key target to cover a large spectrum of GM bacteria. A real-time PCR method to screen for its potential presence as well as a nested-PCR method associated with a sequencing analysis to assess its full-length were developed to target this aadD gene. The performance of these new methods were successfully evaluated in terms of specificity, sensitivity and applicability, allowing their easy implementation in enforcement laboratories. Moreover, the integration of these newly developed methods to our very recently proposed strategy, initially targeting GMM carrying a chloramphenicol resistance gene, allows to drastically increase the detection spectrum of GM bacteria producing fermentation food and feed products. The data generated by the proposed strategy represents therefore a crucial support for the competent authorities, especially to evaluate potential risks for the food and feed safety.

Identification of an unauthorized genetically modified bacteria in food enzyme through whole-genome sequencing.

Recently, the unexpected presence of a viable unauthorized genetically modified bacterium in a commercialized food enzyme (protease) product originating from a microbial fermentation process has been notified at the European level (RASFF 2019.3332). This finding was made possible thanks to the use of the next-generation sequencing technology, as reported in this study. Whole-genome sequencing was used to characterize the genetic modification comprising a sequence from the pUB110 shuttle vector (GenBank: M19465.1), harbouring antimicrobial resistance genes conferring a resistance to kanamycine, neomycin and bleomycin, flanked on each side by a sequence coding for a protease (GenBank: WP_032874795.1). In addition, based on these data, two real-time PCR methods, that can be used by enforcement laboratories, specific to this unauthorized genetically modified bacterium were developed and validated. The present study emphasizes the key role that whole-genome sequencing can take for detection of unknown and unauthorized genetically modified microorganisms in commercialized microbial fermentation products intended for the food and feed chain. Moreover, current issues encountered by the Competent Authorities and enforcement laboratories with such unexpected contaminations and the importance of performing official controls were highlighted.

Genetically Modified Micro-Organisms for Industrial Food Enzyme Production: An Overview.

The use of food enzymes (FE) by the industrial food industry is continuously increasing. These FE are mainly obtained by microbial fermentation, for which both wild-type (WT) and genetically modified (GM) strains are used. The FE production yield can be increased by optimizing the fermentation process, either by using genetically modified micro-organism (GMM) strains or by producing recombinant enzymes. This review provides a general overview of the different methods used to produce FE preparations and how the use of GMM can increase the production yield. Additionally, information regarding the construction of these GMM strains is provided. Thereafter, an overview of the different European regulations concerning the authorization of FE preparations on the European market and the use of GMM strains is given. Potential issues related to the authorization and control of FE preparations sold on the European market are then identified and illustrated by a case study. This process highlighted the importance for control of FE preparations and the consequent need for appropriate detection methods targeting the presence of GMM, which is used in fermentation products.

Strategy for the identification of micro-organisms producing food and feed products: Bacteria producing food enzymes as study case.

Recent European regulations require safety assessments of food enzymes (FE) before their commercialization. FE are mainly produced by micro-organisms, whose viable strains nor associated DNA can be present in the final products. Currently, no strategy targeting such impurities exists in enforcement laboratories. Therefore, a generic strategy of first line screening was developed to detect and identify, through PCR amplification and sequencing of the 16S-rRNA gene, the potential presence of FE producing bacteria in FE preparations. First, the specificity was verified using all microbial species reported to produce FE. Second, an in-house database, with 16S reference sequences from bacteria producing FE, was constructed for their fast identification through blast analysis. Third, the sensitivity was assessed on a spiked FE preparation. Finally, the applicability was verified using commercial FE preparations. Using straightforward PCR amplifications, Sanger sequencing and blast analysis, the proposed strategy was demonstrated to be convenient for implementation in enforcement laboratories.

A new multiplex RT-qPCR method for the simultaneous detection and discrimination of Zika and chikungunya viruses.

OBJECTIVE: The re-emergence and spread of tropical viruses to new areas has raised a wave of concern worldwide. In order to treat patients at an early stage and prevent the diffusion of an outbreak, early diagnosis, and therefore fast and adequate detection, is needed. To this end, a multiplex reverse transcription real-time polymerase chain reaction TaqMan method was designed to detect Zika (ZIKV) and chikungunya (CHIKV) viruses simultaneously. METHODS: Two methods targeting different genome segments were selected from the literature for each virus. These were adapted for high genome coverage and combined in a four-plex assay that was thoroughly validated in-house. The SCREENED tool was used to evaluate the sequence coverage of the method. RESULTS: The full validation approach showed that the new four-plex method allows the specific and sensitive identification and discrimination of ZIKV and CHIKV in routine samples. The combination of two targets per virus allowing almost 100% coverage of about 500 genomes is shown for the first time. CONCLUSIONS: PCR is a reliable user-friendly technique that can be applied in remote areas. Such multiplex methods enable early and efficient diagnosis, leading to rapid treatment and effective confinement in outbreak cases. They may also serve as an aid for surveillance, and the full validation permits easy method-transfer allowing worldwide harmonization.

MinION sequencing technology to characterize unauthorized GM petunia plants circulating on the European Union market.

In order to characterize unauthorized genetically modified petunia, an integrated strategy has been applied here on several suspected petunia samples from the European market. More precisely, DNA fragments of interest were produced by DNA walking anchored on key targets, earlier detected by real-time PCR screening analysis, to be subsequently sequenced using the MinION platform from Oxford Nanopore Technologies. This way, the presence of genetically modified petunia was demonstrated via the characterization of their transgene flanking regions as well as unnatural associations of elements from their transgenic cassette.