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Identification of an unauthorized genetically modified micro organism in food enzyme via total-genome sequencing | 412-79 exam Questions and cheat sheet

A food enzyme (protease) product commercialized on the eu market, used in this look at, turned into these days suspected to be contaminated through unauthorized GMM. On the one hand, starting from DNA extracted from the food enzyme matrix, the presence of bacterial DNA belonging to the Bacillus genus became confirmed by using PCR amplification and Sanger sequencing of the bacterial sixteen S rRNA gene region8 (Fig. 1 step 1). additionally, the potential GM nature of this illness changed into suspected based on a good precise-time PCR sign for the aminoglycoside adenyltransferase (aadD) gene conferring a resistance to each kanamycin (KanR) and neomycine (NeoR) (GenBank: M19465.1) (Fig. 1 step 1)10. The presence of the whole-size size of this aadD gene become as a result tested through nested-PCR combined with Sanger sequencing, highlighting potential fitness and environmental hazards associated to this food enzyme product in light of AMR acquisition concerns9,10. having said that, importantly, a conceivable bacterial pressure become remoted from the proven food enzyme product8. This bacterial strain was subjected to the equal analysis as above utilized on DNA extracted from the meals enzyme matrix. similarly to the outcomes accompanied the usage of DNA extracted from the food enzyme matrix, this bacterial pressure was proven to belong to the Bacillus genus8 as well as to elevate the aadD gene (Fig. 1 step 2, desk 1, Supplementary file 1). The presence of a possible GM Bacillus pressure in the demonstrated trial changed into subsequently strongly suspected, but, these effects were insufficient to absolutely prove the presence of this GMM and represent its linked genetic changes.

determine 1

Schematic illustration of the workflow, composed of three leading successive steps, utilized on the proven food enzyme product, allowing to establish unauthorized GMM by using entire-genome sequencing (WGS) and due to this fact to Excellerate adventure-specific precise-time PCR methods. (1) DNA extracted from the FE guidance became validated for the presence of bacterial DNA as neatly as the presence of AMR genes commonly harboured with the aid of GMM used to supply FE. (2) dwelling microbial traces, past remoted from the FE education, have been validated for the presence of bacterial DNA and subsequent determination of their genus/species in addition to for the presence of AMR genes generally harboured by using GMM used to provide FE. (three) The bacterial lines recognized in (2) as carrying AMR genes turned into characterized by means of a WGS approach using a de novo assembly analysis with the intention to reveal the presence of a achievable unauthorized GMM within the established FE coaching. With the generated sequences, precise-time PCR methods selected to this GMM were developed to be used with the aid of enforcement laboratories.

table 1 Oligonucleotides used for PCR-based mostly strategies. GMM identification the use of WGS

the use of an Illumina MiSeq system (250 bp paired-end reads), WGS utilized on DNA from the bacterial pressure isolated from the food enzyme product (Fig. 1 step 3, Supplementary file 2) generated 714,637 paired-conclusion uncooked reads. Following examine trimming, 589,817 extraordinary reads (normal Phred rating of 37) were retained to operate a de novo meeting, enabling to generate 430 contiguous sequences (contigs) of which forty seven were longer than 1,000 bases with a ok-mer coverage of as a minimum 10x. Contig sizes ranged from 56 bp to 457,195 bp, with an N50 price of 291,658.

On the one hand, the generated contigs introduced a correspondence to the Bacillus genus, and, greater precisely, relatively to the B. velezensis species (RefSeq: NZ_CP011937.1) instead of the expected B. subtilis species that became labelled as being the producer organism of the commercialized neutral protease. This identification became in line with three observations. in the beginning, when using the assembly for typing against the B. subtilis MLST schema hosted with the aid of the PubMLST.org net-based platform, a perfect healthy to sequence category a hundred and forty turned into acquired for which handiest a single isolate become existing in the database (PubMLST: ATCC 12321) annotated as the species B. velezensis. Secondly, a okay-mer based mostly classification of sequencing reads in opposition t an in-house dump of all comprehensive genomes in the RefSeq Microbial Genomes database indicated the presence of B. velezensis (Supplementary file three). Thirdly, this identification turned into demonstrated through performing a read mapping analysis to the NCBI representative B. velezensis reference genome sequence (RefSeq: NZ_CP001937.1), with a median depth and breadth of insurance of respectively 58x and ninety four.58% (Supplementary file four). B. velezensis species isn't listed by way of EFSA (2018) as being used in the food and feed business to produce food and feed additives, enzymes and flavourings supposed for the ecu market30. besides the fact that children, this species, for which the wild-classification is harmless for human and closely related to B. amyloliquefaciens and B. subtilis, has in the past been described as tremendously beneficial for producing enzymes, together with proteases, for the agro-industrial sector31,32,33,34,35,36.

then again, the generated contigs have been blasted against the aadD gene, conferring KanR and NeoR, that turned into earlier detected by using actual-time PCR as well as nested-PCR followed by Sanger sequencing analysis10 (table 1, Supplementary file 1). A contig of 349,285 bp with a k-mer coverage of 59.434x become identified as harbouring the targeted AMR gene (Fig. 2, Supplementary file 5). in an effort to identify the putative transgenic insertion, the regions flanking this AMR gene had been then characterized and compared to the reference genome of B. velezensis (RefSeq: NZ_CP011937.1). in the reference genome, a place of 2,385 bp from place 2,460,164 to 2,462,548, with >ninety nine% sequence id, composed of a gene coding for a protease (GenPept: WP_032874795.1; RS12020 in Fig. 2) as well as a part of a gene coding for an acetyltransferase (GenPept: WP_032874793.1; RS12025 in Fig. 2), become changed with the aid of a fraction of 9,141 bp within the genome of the isolated bacterial strain containing the area of 2,385 bp in reproduction. when you consider that the established food enzyme product became commercialized as a protease, the duplication of this vicinity can for this reason be explained through the goal of the producers to enhance protease yield during the production procedure. Between these duplicated areas, a sequence of 4,102 bp, with a question coverage and id of 100%, matching to the pUB110 shuttle vector (GenBank: M19465.1) harbouring the aadD gene (GenBank: AAA88361.1), conferring KanR and NeoR, that become past recognized by way of actual-time PCR and nested-PCR (Fig. 1), and the ble gene (RefSeq: NG_047557.1), conferring a resistance to bleomycin (BleoR) turned into characterised (Fig. 2, Supplementary file 5). This pUB110 shuttle vector, originating from Staphylococcus aureus, and the recognized AMR genes had been up to now reported as being enormously used in GMM producing bacterial fermentation products in the food and feed chain, principally for preference of lines of interest10,37. in addition, the accompanied left and appropriate transgene flanking regions of the inserted fragment of 9,141 bp as smartly as the left and correct transgene flanking regions of the pUB110 shuttle vector had been Tested with the aid of PCR followed by using Sanger sequencing (Supplementary files 5,6). in keeping with all these consequences, the presence of a genetic modification selected to a attainable GMM within the commercialized meals enzyme product was hence proven. These results, communicated to the Belgian Federal company for the protection of the meals Chain, have resulted in the RASFF 2019.3332 notification at the ecu level.

determine 2

Schematic illustration of the recognized transgenic insertion. The pUB110 shuttle vector (eco-friendly) harbours the aadD gene conferring a resistance to kanamycin (KanR) (red) and the ble gene conferring a resistance to bleomycin (BleoR) (yellow). Blue rectangles represent annotated genes on the reference genome. The vicinity indicated in orange includes a gene coding for a protease (RS12020) and a part of a gene coding for an acetyltransferase (RS12025). The latter, indicated via a small dark red rectangle within the GM consists out of a full (RS12025) and interrupted (RS12025a) replica. The crimson area is exciting within the wild-class whereas this crimson area is duplicated, on each side of the pUB110 shuttle vector, in the GMM. The dark and hatched rectangles indicate the areas focused by the left (L) and appropriate (R) event-certain precise-time PCR strategies developed and validated during this analyze.

related to the bioinformatics methodology, in comparison to a examine-mapping evaluation, a de novo meeting evaluation was probably the most primary strategy to identify and symbolize an unknown and unauthorized GMM for 2 factors. originally, no reference sequence is required, representing an talents e.g. within the existing analyze because of the unavailability of a reference sequence for the identified GMM. This approach is additionally positive when the species id of the GMM host is difficultly identifiable, as exemplified in this examine with the Bacillus strain8. Secondly, through reconstructing a contig containing the transgenic insertion within the wild-class B. velezensis genome, an unnatural affiliation of sequence elements may well be inferred, presenting effective proof of the presence of a GMM. without an purchasable reference sequence for a particular GMM, a read-mapping evaluation cannot provide this type of essential information. indeed, handiest the presence of sequences belonging both to the pUB110 shuttle vector or to B. velezensis could then had been validated, but no link between the pUB110 shuttle vector and B. velezensis might have been centered (Supplementary file four).

building of experience-certain true-time PCR methods based on WGS data

in keeping with characterization of the transgenic insertion into B. velezensis, two adventure-certain real-time PCR methods had been developed and validated, permitting to specifically target cost- and time-effectively the unauthorized GMM found in the current study (Fig. 1 step three). These two experience-selected methods had been designed to cowl either the left or the correct transgene flanking region of the inserted pUB110 shuttle vector (Fig. 2; table 1; Supplementary file 5). For each and every actual-time PCR method, an amplicon with the expected size and sequence turned into acquired (Supplementary file 7). The performance of these actual-time PCR methods turned into then investigated.

First, the specificity of these actual-time PCR strategies was demonstrated the usage of, as high quality control, DNA from the isolated GM B. velezensis RASFF 2019.3332 strain as well as, as negative controls, DNA from eighty-5 wild-type microbial traces commonly used to provide microbial fermentation products9,10, DNA from six different wild-classification B. velezensis traces, DNA from the vitamin B2-producing GM B. subtilis RASFF 2014.1249 pressure, DNA from plant (Zea mays) and DNA from human. As expected, these event-certain actual-time PCR methods introduced a good sign only for the tremendous control, confirming their specificity (table 2). second, the sensitivity of those precise-time PCR strategies changed into assessed the use of DNA from the GM B. velezensis RASFF 2019.3332 stress at different estimated full genome copy numbers (6 × 106, 6 × 104, 6 × 102, 60, 12, 6, 1, 0.1 and nil) (desk 3). For both precise-time PCR strategies, a favorable sign turned into accompanied at as little as one estimated full genome reproduction, demonstrating their high sensitivity. Third, the applicability of these real-time PCR strategies turned into demonstrated the usage of DNA from the commercialized meals enzyme product through which the GM B. velezensis RASFF 2019.3332 pressure (sample n°1) changed into detected as well as a commercialized vitamin B2 feed additive product (RASFF 2014.1249) (sample n°2). As anticipated, both precise-time PCR methods presented a good signal for the pattern n°1 and a bad sign for the pattern n°2 (Supplementary file 6). in accordance with all these effects, the two proposed experience-specific real-time PCR strategies were evaluated as specific, sensitive and relevant, permitting enforcement laboratories to easily target the GM B. velezensis RASFF 2019.3332 pressure in commercialized microbial fermentation items. If imperative, following to further optimisation and validation steps, these real-time PCR methods may be mixed into a duplex assay.

desk 2 listing of wild-category microorganisms used for specificity evaluation of the actual-time PCR methods. The presence and absence of amplification are respectively symbolized by using “+” and “-”. For each outcome, the scan changed into carried out in reproduction. DNA from the GM Bacillus subtilis (RASFF 2014.1249) stress, ninety-one wild-category microbial lines, plant and animal have been used as negative manage. DNA from the GM Bacillus velezensis (RASFF 2019.332) stress changed into used as superb control. desk 3 Sensitivity evaluation of actual-time PCR strategies. For each and every established DNA awareness from the GM Bacillus velezensis RASFF 2019.3332 strain, the corresponding estimated full genome copy quantity is indicated. The presence and absence of amplification are respectively symbolized by means of “+” and “−”. For each and every outcomes at each DNA concentration, the experiment became carried out in quadruplicate. From 25 to 0.0000025 ng, each and every replicate generated a positive sign. The capability of the followed Cq are indicated between brackets. From 0.00000025 to 0 ng, every replicate generated a poor signal.

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