Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction

Background To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. Results In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation. In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Conclusion Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetecte

Comparison and transfer testing of multiplex ligation detection methods for GM plants

<p>Abstract</p> <p>Background</p> <p>With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.</p> <p>Results</p> <p>Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands).</p> <p>Conclusions</p> <p>From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible.</p

Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models.

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies

Development of a multiplex DNA-based traceability tool for crop plant materials

The authenticity of food is of increasing importance for producers, retailers and consumers. All groups benefit from the correct labelling of the contents of food products. Producers and retailers want to guarantee the origin of their products and check for adulteration with cheaper or inferior ingredients. Consumers are also more demanding about the origin of their food for various socioeconomic reasons. In contrast to this increasing demand, correct labelling has become much more complex because of global transportation networks of raw materials and processed food products. Within the European integrated research project ‘Tracing the origin of food’ (TRACE), a DNA-based multiplex detection tool was developed—the padlock probe ligation and microarray detection (PPLMD) tool. In this paper, this method is extended to a 15-plex traceability tool with a focus on products of commercial importance such as the emmer wheat Farro della Garfagnana (FdG) and Basmati rice. The specificity of 14 plant-related padlock probes was determined and initially validated in mixtures comprising seven or nine plant species/varieties. One nucleotide difference in target sequence was sufficient for the distinction between the presence or absence of a specific target. At least 5% FdG or Basmati rice was detected in mixtures with cheaper bread wheat or non-fragrant rice, respectively. The results suggested that even lower levels of (un-)intentional adulteration could be detected. PPLMD has been shown to be a useful tool for the detection of fraudulent/intentional admixtures in premium foods and is ready for the monitoring of correct labelling of premium foods worldwide

Towards a Multiplex Cereal Traceability Tool Using Padlock Probe Ligation on Genomic DNA

Current EU regulations on the protection of products with certain characteristics (geographical indications and designations of origin) aim to ensure fair competition for producers and increased consumers' trust. Within the European integrated research project TRACE analytical methods are being developed to allow the maintenance of specific regulations for PGIs (products of protected geographical indication) and PDOs (products of designated origin). An example within the project is the PGI wheat variety Farro della Garfagnana. Aim of the research was to develop amethod to establish the purity of Farro della Garfagnana DNA in in complex cereal mixtures. The combined approach of padlock probe ligation and multiplex microarray detection can identify possible admixtures. One undesired 'contaminant' for Farro della Garfagnana is common bread wheat (Triticum aestivum), containing the BBAuAuDD genome. Since Farro harbours the BBAuAu genome, absence of the D-genome rules out the presence of bread wheat. The current detection limit of this multimethod is at least 2.5 % bread wheat in Farro.JRC.DDG.D.2-Reference material

Comparison of the effects of digested white and yellow onion extracts on mRNA expression in human Caco-2 cells <i>in vitro</i> to that in rat intestine <i>ex vivo</i>.

<p>First, 429 genes were selected that were >1.5 fold up or downregulated in ≥3 of 9 arrays of the Caco-2 exposure as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160719#pone.0160719.g001" target="_blank">Fig 1A</a>. Thereafter, the mRNA expression data of the same genes for the rat intestine exposure were added, which was followed by unsupervised hierarchical clustering. WO_Dig or YO_Dig: White onion and yellow onion digested.</p

Unsupervised hierarchical clustering of genes altered by exposure to white onion or yellow onion digest.

<p>Genes were selected on > 1.5-fold up- or downregulation by WOd (WO_Dig) or YOd (YO_Dig) in at least 3 out of 6 (Caco-2 and rat intestine) or 7 (SISP) arrays. This resulted in 429, 766 and 713 genes for heatmaps of human Caco2 cells (A), rat intestine slices (B) and the porcine SISP model (C), respectively. Red: upregulation, green: downregulation, black: not affected. A ≥ 2-fold up- or downregulation obtained a maximal colour. The expression data for the three models (in 2log values of onion digest vs. saline digest) are provided are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160719#pone.0160719.s001" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160719#pone.0160719.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160719#pone.0160719.s003" target="_blank">S3</a> Tables.</p

Overlap in onion-induced differentially expressed genes detected in human Caco-2 model, rat slices and pig SISP.

<p>A) Of 9,140 genes that were expressed in all three models, the overlap in onion-induced differential gene expression was determined (raw/unadjusted p-value<0.05 and FC > 1.5 or < 0.66). Expression of overlapping genes changed in the same direction (up- or downregulated). B) Overlap in differentially expressed genes (raw/unadjusted p-value<0.05) between human Caco-2 and pig SISP. C) Overlap in differentially expressed genes (raw/unadjusted p-value<0.05) between human Caco-2 and rat slices. Dark red and green indicates differential gene expression with fold changes >1.5 and <0.667, light red en green indicates fold changes >1.2 and <0.833. N/A; did not meet the 'flooring' criteria (low/no expression), so excluded from further analyses. * no homologues found in pig, only related genes (Sulfiredoxin-1-like).</p

Onion-induced Keap1-Nrf2 pathway activation in human Caco-2 model, rat slices and pig SISP.

<p>Schematic visualisation of the Keap1-Nrf2 pathway, with upstream regulators and downstream target genes of Nrf2. Red indicates upregulation of the genes, green indicates downregulation. The letters next to the genes indicate the model(s) in which genes are affected: H: human Caco-2, R: rat slices, P: pig SISP.</p