BACKGROUND
riboflavin  is necessary for basic cell metabolism but is not synthetized by higher animals and needs to be ingested.
therefore, the vitamin is commercialized for nutritional use in the fortification of various food and feed products.
it is also commonly used as a colorant in food such as ice cream, cheeses, mayonnaises and sauces, and as a medical identification aid, because of its intense yellow color .
at the present time, fermentation using microbiological processes is widely applied to commercially produce riboflavin  <cit>  and genetically modified  bacillus  subtilis overproducing riboflavin is one of the main organism currently used for riboflavin’s industrial production .
one common genetic method used to enhance riboflavin production derived from fermentation by gm b. subtilis, is to clone the riboflavin biosynthesis operon  from b. subtilis under the control of a strong constitutive promoter into  plasmids  .
in the framework of eu legislation  <cit> , companies wanting to market in the eu a specific additive produced by genetically modified microorganisms , like vitamin b <dig>  need to submit an application that will be evaluated by the european food safety authority .
this will result in a scientific opinion concerning the safety and the efficacy of the product efsa  <cit> .
according to the efsa guidance, food and feed additives produced by gmms intended for human and animal consumption must be pure.
this means that both gmms  and newly introduced genes should have been removed from the final product  <cit> .
this is especially important for safety evaluation as the amount of recombinant residual host cell dna as well as the risk of gene transfer need to be assessed.
based on this information, efsa will be able to deliver its scientific opinion concerning the product, including whether or not the production strain or its recombinant dna was detected in the final product .
according to eu legislation  <cit> , labelling is not required for vitamins and additives produced by a gmm because the producer strain  and its components, including dna, are no longer present in the commercialized product as the final product should be carefully purified, not containing any residual gmm material.
therefore, unlike for plant gmos, the companies do not have to provide an event-specific method to trace the gmo in food and feed products on the eu market and there is no control by enforcement laboratories, for the correctness of the labelling.
it is assumed that the company bringing the gmm-derived riboflavin to the market has verified the absence of the gmm or its recombinant dna in the final product, so that only “pure” product can be found on the eu market.
however, very recently, it was demonstrated that gmm-contamination in feed additives could occur as a viable gm -b. subtilis was found in an imported lot of vitamin b <dig> feed additive placed on the eu market.
this has led in september  <dig> to a notification for unauthorized gmo  in the european rapid alert system for food and feed .
confronted with this rasff, as no official method is available for detecting this gmm in food and feed, two methods previously developed for other purposes have been proposed and used by enforcement laboratories controlling the gmo-content in food and feed at the eu market.
the first one allows identifying the presence of b. subtilis strains and includes a pcr amplification of the 16s rrna gene followed by its sequencing  <cit> .
this method is not especially convenient and is more laborious.
indeed, it requires sequencing in addition to the pcr analysis and this is not commonly used by the routine laboratories in gmo analysis.
the second method allows detecting the presence of recombinant dna.
it is a real-time pcr method that was used as an internal amplification control targeting the plasmid puc <dig>  commonly used to construct genetically modified b. subtilis.
this method was originally developed in duplex with a qpcr method for the detection of pathogenic yersinia enterocolitica in food samples  <cit> .
it targets a standard vector construct , and consequently has a large spectrum of targets commonly used in many gmm constructs.
moreover, traces of such kind of recombinant dna could be found in mixes containing optimized taq polymerase enzyme used for pcr.
therefore, these  <dig> methods both provide ambiguous results in the detection of gm-b.
subtilis overproducing riboflavin.
there is a need to develop a faster, specific method targeting this particular gm-strain to be put at the disposition of the enforcement laboratories.
in order to develop such a specific method, the gm-b.
subtilis overproducing riboflavin was isolated from three different samples of imported vitamin b <dig> feed additive and collected by the french competent authorities in the framework of the rasff 2014– <dig>  next generation sequencing  was performed on the genomic dna extracted from one of the isolates.
the sequencing reads were de novo assembled and genome annotation was performed on the contig sequences  <cit> .
however, ngs data analysis often requires appropriate tools, involving bioinformatics expertise which is not always present in the average enforcement laboratory.
this hampers the use of this technology to easily obtain critical sequence information in order to be able to develop a specific qpcr detection method.
in this study, the dna sequences previously generated by the ngs approach  <cit>  were used to develop a taqman® qpcr method targeting the junction between the b. subtilis riboflavin operon and the vector used to construct this gm-strain.
different performance criteria of the developed qpcr method such as specificity, sensitivity, pcr efficiency and repeatability were evaluated according to the gmo guidelines  <cit> .
finally, the qpcr method was assessed directly on the gdna extracts from the three different samples of imported vitamin b <dig> feed additive and collected by the french competent authorities.
the method proposed in the present study provides a crucial tool for identifying specifically and rapidly the gm-b.
subtilis overproducing riboflavin firstly detected in the imported vitamin b <dig> feed additive.
moreover, it illustrates that ngs data can be used very easily, without specific bioinformatics expertise, in order to obtain crucial information to develop specific taqman® qpcr methods, to detect unknown and unauthorized gmo in food and feed.
RESULTS
sequence analysis to identify the junction and design of the gm-specific qpcr assay
the first step in the development of the specific qpcr assay consisted of identifying the junction of the gm-insert into the bacillus subtilis gm-strains extracted from vitamin b <dig> samples 2014– <dig>   <cit> .
as elaborated in methods, a blast study of the contigs of the previously published sequence of the gm-bacillus subtilis 2014– <dig> isolated from vitamin b <dig>  <dig> %   <cit>  was used to identify the contigs containing the riboflavin biosynthesis operon ribgbah, with a focus on locating the riba gene as it has been previously reported that additional expression of this gene encoding the rate limiting enzyme in riboflavin synthesis increases riboflavin synthesis even more as compared to only overexpression of ribgbah  <cit> .
the overlapping contigs contig <dig>  and contig <dig>  were retrieved as containing genes having  <dig> % similarity with b. subtilis subsp.
subtilis str.
<dig> genes ribh, ribba, ribe and ribd, involved in riboflavin biosynthesis  <cit> , as it could also be deduced from the annotation of the gm-b.
subtilis genome sequence   <cit>  .
using the publicly available genome sequence of b. subtilis subsp.
subtilis str.
<dig>  and a blast analysis, contig <dig> was identified as the other contig adjacent to contig <dig> .
however, a gap of  <dig> basepairs between contig <dig> and contig <dig> was observed when comparing these contig sequences to the b. subtilis subsp.
subtilis str.
<dig> genome sequence  .
pcr and sequence analysis was used to confirm that the genomic regions present on both contigs are indeed adjacent in the gm-bacillus genome .
further investigation of contig <dig> and contig <dig> revealed the presence of a region with  <dig> % similarity to the psm <dig>  or pbt <dig>  plasmid .
it needs to be further investigated whether these plasmid sequences are integrated in the gm-bacillus genome.
nevertheless, this vector has probably been used to construct the gm-strain.
therefore, the junctions between the non-naturally present vector sequences and the bacillus region containing the riboflavin biosynthesis genes  were to be targeted by the event-specific taqman® qpcr assay.
the gm-cassette junctions were found around position  <dig> in the contig <dig> and around position  <dig> in the contig <dig> .
primer pairs and probes have been designed at these positions and tested on the gm-bacillus subtilis 2014– <dig> .
one of the assays targeting the junction on the contig <dig> was kept for further evaluation .
this assay, called the vitb2-ugm qpcr assay, was then evaluated for its specificity.fig.
<dig> amplicon, primers and probe sequences of the vitb2-ugm qpcr assay, position of the junction gm-plasmid-insert.
green: similarity with plasmid psm <dig> and pbt <dig>  light blue: “insert” – similarity with bacillus subtilis subsp.
subtilis str.
<dig> sequences ; striped blue box: gap of  <dig> basepairs between contig <dig> and contig <dig>  sequence obtained by pcr followed by sequencing; underlined: primers of the vitb2-ugm qpcr assay, bold: probe of the vitb2-ugm qpcr assay; the size and orientation of the contigs has been indicated at the top, as also the position of the junctions in the respective contigs; genes located on the respective contigs are indicated with green and light blue arrows using annotation of b. subtilis strain 2014– <dig>   – genes encoding following proteins: bin <dig> - putative transposon tn <dig> dna-invertase; hyp – hypothetical protein; reps - protein reps; copr - plasmid copy control protein copr; kix <dig> : accumulation-associated protein; hyp - hypothetical protein; tcpc - conjugative transposon protein tcpc; ypui_ <dig> - part of putative protein ypui_2; scpb - segregation and condensation protein b; scpa- <dig> - segregation and condensation protein a; ypuf - putative protein ypuf; ribt_ <dig> - protein ribt; ribh_ <dig> -  <dig> -dimethyl-8-ribityllumazine synthase; ribba_ <dig> = gtp cyclohydrolase-2; ribe_ <dig> - riboflavin synthase; ribd_1–5-amino-6-uracil reductase; ypud - putative protein ypud; sips - signal peptidase i s; ypzj - putative protein ypzj; hyp - hypothetical protein; ypub - putative protein ypub; ypzd - spore germination protein-like protein ypzd; hyp - hypothetical protein; hyp - hypothetical protein; kix <dig>  - n-acetylmuramoyl-l-alanine amidase domain-containing protein; hyp - hypothetical protein; hyp – hypothetical protein
determination of vitb2-ugm qpcr assay’s specificity
the specificity test allows testing the inclusivity and exclusivity of the vitb2-ugm qpcr assay.
the experimental design involved  <dig> bacillus subtilis gm-strains overproducing riboflavin that were extracted from three samples of imported vitamin b <dig>  <dig> %,  <dig> non-target strains representing  <dig> species belonging to  <dig> genera, a ctd  and an ntc .
the non-target microorganisms to test the exclusivity were chosen among taxonomically closely related  and not closely related  bacteria that can be present in the environment and in food and feed matrices  <cit> .
in feed/food additives, normally there should not be any bacteria present, as elaborated above.
however, some species also known to be used as gmm were included in the specificity test .
the vitb2-ugm qpcr assay amplified  <dig> %  of the tested gm-b.
subtilis strains overproducing riboflavin and none  of the non-target samples nor the no template control  nor the diluent dna  .
thus, the detection of the gm-bacillus subtilis 2014– <dig> using the vitb2-ugm qpcr assay is  <dig> % specific for the gm-strain.
this was somehow expected as the vitb <dig> ugm qpcr assay is a construct specific assay  and non-naturally present sequences.
the presence of this junction is not expected in wild-type bacteria, no matter what their origin or phylogenetic relationship is.
the sequence of the amplicon was determined and corresponded to the expected one .table  <dig> selectivity assessment of the vitb2-ugm qpcr assay for detection of the gm-bacillus subtilis 2014– <dig> overproducing riboflavin
a
cq: cq value obtained with the vitb2-ugm qpcr assay under the conditions described in methods; n/a: no amplification; a: reference numbers of b. subtilis strains used: w04-s <dig>  e07-s <dig>  e08-s <dig>  w05-s <dig>  w10-s <dig>  e11-s <dig>  e12-s <dig>  e14-s <dig>  e16-s <dig>  w13-s <dig>  e23-s <dig>  w16-s <dig>  e28-s <dig>  e33-s <dig>  e34-s <dig>  e38-s <dig>  w24-s <dig>  si <dig>  si <dig>  sub <dig>  sub <dig>  sub <dig>  bnbs <dig>  bnbs6; scl: service commun des laboratoires, illkirch-graffenstaden, france; ucl: food and environmental microbiology, earth and life institute, faculty of bioscience engineering, université catholique de louvain, louvain-la-neuve, belgium; iph-fp: foodborne pathogens service, scientific institute of public health, brussels, belgium; salm-nrc: belgian salmonella and shigella national reference center, scientific institute of public health, brussels, belgium; iph-qml: quality of medical laboratories, scientific institute of public health, brussels, belgium; list-nrc: belgian listeria national reference center, scientific institute of public health, brussels, belgium
determination of vitb2-ugm qpcr assay’s dynamic range and pcr efficiency
the dynamic range of a qpcr assay is the range of concentrations where the assay performs linearly.
this was assessed for the vitb2-ugm qpcr assay by the analysis in duplicate of a serial dilution of gdna  of the gm-bacillus subtilis strain 2014– <dig> overproducing riboflavin  <cit> .
in addition, this analysis allowed for the assessment of the coefficient of determination  and the pcr efficiency .
the coefficient of determination  is an indicator of how well the data fit the linear regression curve.
the vitb2-ugm qpcr assay performed linearly between  <dig> and  <dig>  genomic copies as its r2-value, i.e.
<dig> , exceeds the required  <dig>   <cit>  .
from the dynamic range analysis, the pcr efficiency  was calculated.
the vitb2-ugm qpcr assay displayed a pcr efficiency of  <dig>  %, thereby corresponding to the accepted limits fixed for qpcr qualitative method used to detect gmo of 80– <dig> % as well as to the ones used to quantify gmo of 90– <dig> %  <cit> .fig.
<dig> dynamic range, coefficient of determination  and pcr efficiency  of the developed vitb2-ugm qpcr assay.
data were obtained from two replicates at each concentration expressed in estimated copy number of genome
determination of vitb2-ugm qpcr assay sensitivity and repeatability
the sensitivity test was performed to determine the lod of the vitb2-ugm qpcr assay.
the lod is defined as the lowest concentration of an analyte giving a positive result with a probability of  <dig> %  <cit> .
the lod of the vitb2-ugm qpcr assay was determined to be between  <dig> and  <dig> copies  complying with the requirement “below  <dig> genomic copies”  <cit> .
the r-values of the cq-values ranged between  <dig>  to  <dig>  cq.
the rsdr-values of the cq-values were between  <dig>  to  <dig>  % .
there is no limit fixed for these criteria for qualitative qpcr methods  <cit> .table  <dig> limit of detection determination and repeatability of cq-values of the vitb2-ugm qpcr assay
average, standard deviation and percentage of positive signal of cq-values and statistical analysis of the cq values obtained at each dilution point .
n/a: no amplification; na: not applicable.
dbc: dilution buffer control
test of the vitb2-ugm qpcr assay on vitamin b <dig>  <dig> % feed additive samples
to verify if the developed vitb2-ugm qpcr assay targeting the gm-bacillus subtilis 2014– <dig> overproducing riboflavin is able to detect this gm strain in real-life samples, the developed qpcr assay has been tested on gdna extracts from the three samples of imported vitamin b <dig>  <dig> % feed additive and containing these gm-bacillus strains.
the dna extracted from each of the samples gave a clear signal when using the vitb2-ugm qpcr assay at a cq-value around  <dig> for  <dig> times dilutions and around  <dig>  for  <dig> times dilutions .table  <dig> test of vitb2-ugm qpcr assay on vitamin b <dig>  <dig> % samples
na not applicable, n/a no amplification
this demonstrates that the vitb2-ugm qpcr assay can be used to detect the gm-bacillus strain in real-life samples.
CONCLUSIONS
as no method was available to specifically detect the presence of an unauthorized gmo consisting of a gm bacillus subtilis bacterium overproducing vitamin b <dig> found in a feed additive , in the present study a new taqman® qpcr method specifically detecting this gm vitamin b <dig> overproducing bacterium was designed.
to be able to develop the vitb2-ugm qpcr assay, critical sequence information was retrieved and used from massive sequence data previously obtained by an ngs approach  <cit> .
to analyse these data, a very simple strategy based on the common blast tools, that can be used by any enforcement lab without profound bioinformatics expertise, was successfully followed.
this allowed to design a specific qpcr method targeting the junction between inserted endogenous riboflavin biosynthesis genes  and non-naturally present sequences .
to guarantee the high-standard quality of the vitb2-ugm method, acceptance parameters like specificity, sensitivity, pcr efficiency and repeatability commonly used to validate a qualitative qpcr method for the detection of gmo were evaluated  <cit> .
the method complies with the acceptance criteria for all of the assessed parameters.
in addition, to further assess the applicability of the newly developed method not only for isolated strains but also for feed products, the vitb2-ugm method was successfully tested on gdna immediately extracted from the feed additive from where the gm-strains were isolated.
this confirms its effective use in routine analysis for reliable detection by enforcement laboratories of the gm- b. subtilis bacterium overproducing vitamin b <dig> .
in the future, this specific method can be used to screen feed and food products regarding this specific gm-bacterium overproducing vitamin b <dig>  however, this method might have a broader application, as vitamin b <dig> is used in a wide range of products present at both the food and feed market  <cit> .
moreover, this vitamin is not a unique example of additive produced by gmm.
indeed, gmm are used to produce other vitamins, additives and processing agents for the food industry  <cit> .
companies are responsible for ensuring that their food and feed put at the market are safe, pure, and that it complies with legislation on food and feed additives and regulation on reducing or eliminating human health risks caused by possible contaminants.
however, in view of the rasff demonstrating the presence of an unauthorized gm  contaminant in feed, the intensive use of gmm to synthetize food and feed additives, as well the possible import of these products in eu, the question is raised if a more systematic survey of the eu market for gmm food and feed contaminants in additives is not necessary.
hereto, our study underlines that when this ngs strategy is applied on unknown and unauthorized gmm, it delivers a massive amount of information that can be easily used by enforcement laboratories without specific bioinformatics expertise for the development of specific qpcr methods targeting these ugm in the food and feed market.

 riboflavin is necessary for basic cell metabolism but is not synthetized by higher animals and needs to be ingested. therefore, the vitamin is commercialized for nutritional use in the fortification of various food and feed products. it is also commonly used as a colorant in food such as ice cream, cheeses, mayonnaises and sauces, and as a medical identification aid, because of its intense yellow color. at the present time, fermentation using microbiological processes is widely applied to commercially produce riboflavin and genetically modified bacillus subtilis overproducing riboflavin is one of the main organism currently used for riboflavin’s industrial production. one common genetic method used to enhance riboflavin production derived from fermentation by gm b. subtilis, is to clone the riboflavin biosynthesis operon from b. subtilis under the control of a strong constitutive promoter into plasmids. in the framework of eu legislation, companies wanting to market in the eu a specific additive produced by genetically modified microorganisms, like vitamin b need to submit an application that will be evaluated by the european food safety authority. this will result in a scientific opinion concerning the safety and the efficacy of the product efsa. according to the efsa guidance, food and feed additives produced by gmms intended for human and animal consumption must be pure. this means that both gmms and newly introduced genes should have been removed from the final product. this is especially important for safety evaluation as the amount of recombinant residual host cell dna as well as the risk of gene transfer need to be assessed. based on this information, efsa will be able to deliver its scientific opinion concerning the product, including whether or not the production strain or its recombinant dna was detected in the final product. according to eu legislation, labelling is not required for vitamins and additives produced by a gmm because the producer strain and its components, including dna, are no longer present in the commercialized product as the final product should be carefully purified, not containing any residual gmm material. therefore, unlike for plant gmos, the companies do not have to provide an event-specific method to trace the gmo in food and feed products on the eu market and there is no control by enforcement laboratories, for the correctness of the labelling. it is assumed that the company bringing the gmm-derived riboflavin to the market has verified the absence of the gmm or its recombinant dna in the final product, so that only “pure” product can be found on the eu market. however, very recently, it was demonstrated that gmm-contamination in feed additives could occur as a viable gm -b. subtilis was found in an imported lot of vitamin b feed additive placed on the eu market. this has led in september to a notification for unauthorized gmo in the european rapid alert system for food and feed. confronted with this rasff, as no official method is available for detecting this gmm in food and feed, two methods previously developed for other purposes have been proposed and used by enforcement laboratories controlling the gmo-content in food and feed at the eu market. the first one allows identifying the presence of b. subtilis strains and includes a pcr amplification of the 16s rrna gene followed by its sequencing. this method is not especially convenient and is more laborious. indeed, it requires sequencing in addition to the pcr analysis and this is not commonly used by the routine laboratories in gmo analysis. the second method allows detecting the presence of recombinant dna. it is a real-time pcr method that was used as an internal amplification control targeting the plasmid puc commonly used to construct genetically modified b. subtilis. this method was originally developed in duplex with a qpcr method for the detection of pathogenic yersinia enterocolitica in food samples. it targets a standard vector construct, and consequently has a large spectrum of targets commonly used in many gmm constructs. moreover, traces of such kind of recombinant dna could be found in mixes containing optimized taq polymerase enzyme used for pcr. therefore, these methods both provide ambiguous results in the detection of gm-b. subtilis overproducing riboflavin. there is a need to develop a faster, specific method targeting this particular gm-strain to be put at the disposition of the enforcement laboratories. in order to develop such a specific method, the gm-b. subtilis overproducing riboflavin was isolated from three different samples of imported vitamin b feed additive and collected by the french competent authorities in the framework of the rasff 2014– next generation sequencing was performed on the genomic dna extracted from one of the isolates. the sequencing reads were de novo assembled and genome annotation was performed on the contig sequences. however, ngs data analysis often requires appropriate tools, involving bioinformatics expertise which is not always present in the average enforcement laboratory. this hampers the use of this technology to easily obtain critical sequence information in order to be able to develop a specific qpcr detection method. in this study, the dna sequences previously generated by the ngs approach were used to develop a taqman® qpcr method targeting the junction between the b. subtilis riboflavin operon and the vector used to construct this gm-strain. different performance criteria of the developed qpcr method such as specificity, sensitivity, pcr efficiency and repeatability were evaluated according to the gmo guidelines. finally, the qpcr method was assessed directly on the gdna extracts from the three different samples of imported vitamin b feed additive and collected by the french competent authorities. the method proposed in the present study provides a crucial tool for identifying specifically and rapidly the gm-b. subtilis overproducing riboflavin firstly detected in the imported vitamin b feed additive. moreover, it illustrates that ngs data can be used very easily, without specific bioinformatics expertise, in order to obtain crucial information to develop specific taqman® qpcr methods, to detect unknown and unauthorized gmo in food and feed.  sequence analysis to identify the junction and design of the gm-specific qpcr assay the first step in the development of the specific qpcr assay consisted of identifying the junction of the gm-insert into the bacillus subtilis gm-strains extracted from vitamin b samples 2014–. as elaborated in methods, a blast study of the contigs of the previously published sequence of the gm-bacillus subtilis 2014– isolated from vitamin b % was used to identify the contigs containing the riboflavin biosynthesis operon ribgbah, with a focus on locating the riba gene as it has been previously reported that additional expression of this gene encoding the rate limiting enzyme in riboflavin synthesis increases riboflavin synthesis even more as compared to only overexpression of ribgbah. the overlapping contigs contig and contig were retrieved as containing genes having % similarity with b. subtilis subsp. subtilis str. genes ribh, ribba, ribe and ribd, involved in riboflavin biosynthesis, as it could also be deduced from the annotation of the gm-b. subtilis genome sequence. using the publicly available genome sequence of b. subtilis subsp. subtilis str. and a blast analysis, contig was identified as the other contig adjacent to contig. however, a gap of basepairs between contig and contig was observed when comparing these contig sequences to the b. subtilis subsp. subtilis str. genome sequence. pcr and sequence analysis was used to confirm that the genomic regions present on both contigs are indeed adjacent in the gm-bacillus genome. further investigation of contig and contig revealed the presence of a region with % similarity to the psm or pbt plasmid. it needs to be further investigated whether these plasmid sequences are integrated in the gm-bacillus genome. nevertheless, this vector has probably been used to construct the gm-strain. therefore, the junctions between the non-naturally present vector sequences and the bacillus region containing the riboflavin biosynthesis genes were to be targeted by the event-specific taqman® qpcr assay. the gm-cassette junctions were found around position in the contig and around position in the contig. primer pairs and probes have been designed at these positions and tested on the gm-bacillus subtilis 2014–. one of the assays targeting the junction on the contig was kept for further evaluation. this assay, called the vitb2-ugm qpcr assay, was then evaluated for its specificity.fig. amplicon, primers and probe sequences of the vitb2-ugm qpcr assay, position of the junction gm-plasmid-insert. green: similarity with plasmid psm and pbt light blue: “insert” – similarity with bacillus subtilis subsp. subtilis str. sequences; striped blue box: gap of basepairs between contig and contig sequence obtained by pcr followed by sequencing; underlined: primers of the vitb2-ugm qpcr assay, bold: probe of the vitb2-ugm qpcr assay; the size and orientation of the contigs has been indicated at the top, as also the position of the junctions in the respective contigs; genes located on the respective contigs are indicated with green and light blue arrows using annotation of b. subtilis strain 2014– – genes encoding following proteins: bin - putative transposon tn dna-invertase; hyp – hypothetical protein; reps - protein reps; copr - plasmid copy control protein copr; kix: accumulation-associated protein; hyp - hypothetical protein; tcpc - conjugative transposon protein tcpc; ypui_ - part of putative protein ypui_2; scpb - segregation and condensation protein b; scpa- - segregation and condensation protein a; ypuf - putative protein ypuf; ribt_ - protein ribt; ribh_ - -dimethyl-8-ribityllumazine synthase; ribba_ = gtp cyclohydrolase-2; ribe_ - riboflavin synthase; ribd_1–5-amino-6-uracil reductase; ypud - putative protein ypud; sips - signal peptidase i s; ypzj - putative protein ypzj; hyp - hypothetical protein; ypub - putative protein ypub; ypzd - spore germination protein-like protein ypzd; hyp - hypothetical protein; hyp - hypothetical protein; kix - n-acetylmuramoyl-l-alanine amidase domain-containing protein; hyp - hypothetical protein; hyp – hypothetical protein determination of vitb2-ugm qpcr assay’s specificity the specificity test allows testing the inclusivity and exclusivity of the vitb2-ugm qpcr assay. the experimental design involved bacillus subtilis gm-strains overproducing riboflavin that were extracted from three samples of imported vitamin b %, non-target strains representing species belonging to genera, a ctd and an ntc. the non-target microorganisms to test the exclusivity were chosen among taxonomically closely related and not closely related bacteria that can be present in the environment and in food and feed matrices. in feed/food additives, normally there should not be any bacteria present, as elaborated above. however, some species also known to be used as gmm were included in the specificity test. the vitb2-ugm qpcr assay amplified % of the tested gm-b. subtilis strains overproducing riboflavin and none of the non-target samples nor the no template control nor the diluent dna. thus, the detection of the gm-bacillus subtilis 2014– using the vitb2-ugm qpcr assay is % specific for the gm-strain. this was somehow expected as the vitb ugm qpcr assay is a construct specific assay and non-naturally present sequences. the presence of this junction is not expected in wild-type bacteria, no matter what their origin or phylogenetic relationship is. the sequence of the amplicon was determined and corresponded to the expected one.table selectivity assessment of the vitb2-ugm qpcr assay for detection of the gm-bacillus subtilis 2014– overproducing riboflavin a cq: cq value obtained with the vitb2-ugm qpcr assay under the conditions described in methods; n/a: no amplification; a: reference numbers of b. subtilis strains used: w04-s e07-s e08-s w05-s w10-s e11-s e12-s e14-s e16-s w13-s e23-s w16-s e28-s e33-s e34-s e38-s w24-s si si sub sub sub bnbs bnbs6; scl: service commun des laboratoires, illkirch-graffenstaden, france; ucl: food and environmental microbiology, earth and life institute, faculty of bioscience engineering, université catholique de louvain, louvain-la-neuve, belgium; iph-fp: foodborne pathogens service, scientific institute of public health, brussels, belgium; salm-nrc: belgian salmonella and shigella national reference center, scientific institute of public health, brussels, belgium; iph-qml: quality of medical laboratories, scientific institute of public health, brussels, belgium; list-nrc: belgian listeria national reference center, scientific institute of public health, brussels, belgium determination of vitb2-ugm qpcr assay’s dynamic range and pcr efficiency the dynamic range of a qpcr assay is the range of concentrations where the assay performs linearly. this was assessed for the vitb2-ugm qpcr assay by the analysis in duplicate of a serial dilution of gdna of the gm-bacillus subtilis strain 2014– overproducing riboflavin. in addition, this analysis allowed for the assessment of the coefficient of determination and the pcr efficiency. the coefficient of determination is an indicator of how well the data fit the linear regression curve. the vitb2-ugm qpcr assay performed linearly between and genomic copies as its r2-value, i.e., exceeds the required. from the dynamic range analysis, the pcr efficiency was calculated. the vitb2-ugm qpcr assay displayed a pcr efficiency of %, thereby corresponding to the accepted limits fixed for qpcr qualitative method used to detect gmo of 80– % as well as to the ones used to quantify gmo of 90– %.fig. dynamic range, coefficient of determination and pcr efficiency of the developed vitb2-ugm qpcr assay. data were obtained from two replicates at each concentration expressed in estimated copy number of genome determination of vitb2-ugm qpcr assay sensitivity and repeatability the sensitivity test was performed to determine the lod of the vitb2-ugm qpcr assay. the lod is defined as the lowest concentration of an analyte giving a positive result with a probability of %. the lod of the vitb2-ugm qpcr assay was determined to be between and copies complying with the requirement “below genomic copies”. the r-values of the cq-values ranged between to cq. the rsdr-values of the cq-values were between to %. there is no limit fixed for these criteria for qualitative qpcr methods.table limit of detection determination and repeatability of cq-values of the vitb2-ugm qpcr assay average, standard deviation and percentage of positive signal of cq-values and statistical analysis of the cq values obtained at each dilution point. n/a: no amplification; na: not applicable. dbc: dilution buffer control test of the vitb2-ugm qpcr assay on vitamin b % feed additive samples to verify if the developed vitb2-ugm qpcr assay targeting the gm-bacillus subtilis 2014– overproducing riboflavin is able to detect this gm strain in real-life samples, the developed qpcr assay has been tested on gdna extracts from the three samples of imported vitamin b % feed additive and containing these gm-bacillus strains. the dna extracted from each of the samples gave a clear signal when using the vitb2-ugm qpcr assay at a cq-value around for times dilutions and around for times dilutions.table test of vitb2-ugm qpcr assay on vitamin b % samples na not applicable, n/a no amplification this demonstrates that the vitb2-ugm qpcr assay can be used to detect the gm-bacillus strain in real-life samples.  as no method was available to specifically detect the presence of an unauthorized gmo consisting of a gm bacillus subtilis bacterium overproducing vitamin b found in a feed additive, in the present study a new taqman® qpcr method specifically detecting this gm vitamin b overproducing bacterium was designed. to be able to develop the vitb2-ugm qpcr assay, critical sequence information was retrieved and used from massive sequence data previously obtained by an ngs approach. to analyse these data, a very simple strategy based on the common blast tools, that can be used by any enforcement lab without profound bioinformatics expertise, was successfully followed. this allowed to design a specific qpcr method targeting the junction between inserted endogenous riboflavin biosynthesis genes and non-naturally present sequences. to guarantee the high-standard quality of the vitb2-ugm method, acceptance parameters like specificity, sensitivity, pcr efficiency and repeatability commonly used to validate a qualitative qpcr method for the detection of gmo were evaluated. the method complies with the acceptance criteria for all of the assessed parameters. in addition, to further assess the applicability of the newly developed method not only for isolated strains but also for feed products, the vitb2-ugm method was successfully tested on gdna immediately extracted from the feed additive from where the gm-strains were isolated. this confirms its effective use in routine analysis for reliable detection by enforcement laboratories of the gm- b. subtilis bacterium overproducing vitamin b. in the future, this specific method can be used to screen feed and food products regarding this specific gm-bacterium overproducing vitamin b however, this method might have a broader application, as vitamin b is used in a wide range of products present at both the food and feed market. moreover, this vitamin is not a unique example of additive produced by gmm. indeed, gmm are used to produce other vitamins, additives and processing agents for the food industry. companies are responsible for ensuring that their food and feed put at the market are safe, pure, and that it complies with legislation on food and feed additives and regulation on reducing or eliminating human health risks caused by possible contaminants. however, in view of the rasff demonstrating the presence of an unauthorized gm contaminant in feed, the intensive use of gmm to synthetize food and feed additives, as well the possible import of these products in eu, the question is raised if a more systematic survey of the eu market for gmm food and feed contaminants in additives is not necessary. hereto, our study underlines that when this ngs strategy is applied on unknown and unauthorized gmm, it delivers a massive amount of information that can be easily used by enforcement laboratories without specific bioinformatics expertise for the development of specific qpcr methods targeting these ugm in the food and feed market. 
this has demonstrated that a contamination by a gm micro-organism may occur in feed additives and has confronted for the first time,the enforcement laboratories with this type of rasff. the proposed method, provides a crucial tool for specifically and rapidly identifying this unauthorized gm bacterium in food and feed additives by enforcement laboratories. the results were used to design and assess a new taqman® qpcr method, specifically detecting this gm vitamin b overproducing bacterium. in this study, a very simple strategy based on the common blast tools that can be used by any enforcement lab without profound bioinformatics expertise, was successfully used toanalyse the b. subtilis data generated by ngs. however, ngs data analysis often requires appropriate tools, involving bioinformatics expertise which is not alwayspresent in the average enforcement laboratory. this hampers the use of this technology to rapidly obtain critical sequence information in order to be able to develop a specific qpcrdetection method. the vitb2-ugm method also could detect the b. subtilis strain in genomic dna extracted from the feed additive, without prior culturing step. moreover, this work can be seen as a case study to substantiate how the use of ngs data can offer an added value to easily gain access to sequence information needed to develop qpcr methods to detect unknown andunauthorized gmo in food and feed. recently, the presence of an unauthorized genetically modified bacillus subtilis bacterium overproducing vitamin b in a feed additive was notified by the rapid alert system for food and feed. a taqman® qpcr method was developed and tested on isolated bacterial strains and on the feed additive directly. the method complies with eu critical performance parameters for specificity, sensitivity, pcr efficiency and repeatability.
 recently, the presence of an unauthorized genetically modified bacillus subtilis bacterium overproducing vitamin b in a feed additive was notified by the rapid alert system for food and feed. this has demonstrated that a contamination by a gm micro-organism may occur in feed additives and has confronted for the first time,the enforcement laboratories with this type of rasff. as no sequence information of this gmm nor any specific detection or identification method was available, next generationsequencing was used to generate sequence information. however, ngs data analysis often requires appropriate tools, involving bioinformatics expertise which is not alwayspresent in the average enforcement laboratory. this hampers the use of this technology to rapidly obtain critical sequence information in order to be able to develop a specific qpcrdetection method. methods data generated by ngs were exploited using a simple blast approach. a taqman® qpcr method was developed and tested on isolated bacterial strains and on the feed additive directly.  in this study, a very simple strategy based on the common blast tools that can be used by any enforcement lab without profound bioinformatics expertise, was successfully used toanalyse the b. subtilis data generated by ngs. the results were used to design and assess a new taqman® qpcr method, specifically detecting this gm vitamin b overproducing bacterium. the method complies with eu critical performance parameters for specificity, sensitivity, pcr efficiency and repeatability. the vitb2-ugm method also could detect the b. subtilis strain in genomic dna extracted from the feed additive, without prior culturing step.  the proposed method, provides a crucial tool for specifically and rapidly identifying this unauthorized gm bacterium in food and feed additives by enforcement laboratories. moreover, this work can be seen as a case study to substantiate how the use of ngs data can offer an added value to easily gain access to sequence information needed to develop qpcr methods to detect unknown andunauthorized gmo in food and feed. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords identificationevent-specificgmounauthorized gm-bacillus subtilisriboflavinvitamin b2qpcrissue-copyright-statement© the author 2015 