Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR

<div><p>In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and <i>hmg</i> maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.</p> </div

Precision of the duplex ddPCR assay as a function of the target concentration.

<p>MON810 content measured by ddPCR in five series of seven target concentrations. The target MON810 content (3.85%) is indicated by a dotted line. Acceptance criterion for precision is ±25% of the target content (from 2.89% to 4.81%) represented by the dashed lines. Error bars represent the standard deviation of the measured MON810% by ddPCR at each target concentration (five replicates per target concentration).</p

Dynamic range of the ddPCR duplex assay.

<p>Five replicates for each data point. Error bars represent the standard deviation between the five replicates at each target concentration.</p

Summary table of qPCR, ddPCR and cdPCR performance for MON810 detection and quantification.

<p>qPCR: data produced in this study, or obtained from the literature, when indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Burns1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-EuropeanUnionReferenceLaboratoryforGM1" target="_blank">[22]</a></p><p>ddPCR: data produced in this study.</p><p>cdPCR: data produced on a BioMark System (Fluidigm, South San Francisco) using the 12.765 digital arrays (Fluidigm) and obtained from the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Corbisier1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Burns1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Bhat1" target="_blank">[19]</a>.</p><p>Repeatability through the dynamic range: assessed through the coefficient of variation (Cv) of the target copy numbers or the MON810 content between repeats.</p><p>Trueness: assessed through the calculation of the bias between the MON810 content measured and the target MON810 content. * For our study, trueness is indicated only when qPCR and ddPCR results could be compared to a third, independent value (obtained from the CRM provider or proficiency test organizer).</p><p>Time for results/96 well plate: Total time needed from DNA pipetting to the analysis of the results; reaction mixes are already prepared.</p><p>Price/sample if 96-well plate: Price based on material and reagent costs available at NIB, including labor cost.</p><p>N.A.: not evaluated.</p

Gene expression pattern of <i>MKK</i> family in the HR response against PVY.

<p>Cultivar Rywal (HR response, conferred by <i>Ny-1</i> gene) and NahG-Rywal (impaired accumulation of SA) were analysed for whole transcriptome response 1, 3 and 6 days after PVY^N-Wi infection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104553#pone.0104553-Baebler2" target="_blank">[24]</a>. <i>A. thaliana</i> and <i>S. tuberosum</i> PGSC orthologues were assigned to each probe. Log₂ fold changes of PVY in infected vs. mock-inoculated plants are indicated for each time point (1, 3 and 6 dpi). Statistically significant differences (FDR corrected p<0.05) are in bold. Up-regulated values are in blue and down-regulated values are in yellow.</p

Epidermal cells of <i>N. benthamiana</i> expressing translational fusion of StMKK6 with YFP under native promoter.

<p>Leaves were agroinfiltrated when the virus has spread uniformly through the inoculated leaves (8 days after inoculation) and observed after 72 h in two independent experiments. Examples from two plants (left and right panels) are shown. Control of transformation (fluorescent marker without StMKK6 fusion) is in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104553#pone.0104553.s001" target="_blank">Figure S1A</a>. <b>A.</b> Localisation of StMKK6 in mock-inoculated leaves. No fluorescence was observed. <b>B.</b> Localisation of StMKK6 in PVY-inoculated leaves, where the protein accumulates predominantly in nucleus. Additional images of StMKK6 localisation under native promoter are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104553#pone.0104553.s001" target="_blank">Figures S1D and S1E</a>.</p

St<i>MKK6</i> gene expression in development and stress [40].

<p>Data are obtained from the potato eFP browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104553#pone.0104553-Winter1" target="_blank">[39]</a>. <b>A.</b> Tissue and developmental gene expression pattern of St<i>MKK6</i> in double monoploid <i>S. tuberosum</i> Group Phureja (left) and in heterozygous diploid <i>S. tuberosum</i> Group Tuberosum (right). <b>B.</b> Changes in St<i>MKK6</i> gene expression under biotic stress conditions (treatments of leaves with <i>P. infestans</i>, SA analogues acibenzolar-S-methyl and fungal elicitor DL-β-amino-n-butyric acid and wounding) and abiotic stress conditions (treatments of whole plants with salt, heat and hormones cytokinins, 6 benzylaminopurine; gibberellins, GA3; abscisic acid; and auxin, IAA) and control treatments.</p

Involvement of Potato (<i>Solanum tuberosum</i> L.) MKK6 in Response to <i>Potato virus Y</i>

<div><p>Mitogen-activated protein kinase (MAPK) cascades have crucial roles in the regulation of plant development and in plant responses to stress. Plant recognition of pathogen-associated molecular patterns or pathogen-derived effector proteins has been shown to trigger activation of several MAPKs. This then controls defence responses, including synthesis and/or signalling of defence hormones and activation of defence related genes. The MAPK cascade genes are highly complex and interconnected, and thus the precise signalling mechanisms in specific plant–pathogen interactions are still not known. Here we investigated the MAPK signalling network involved in immune responses of potato (<i>Solanum tuberosum</i> L.) to <i>Potato virus Y</i>, an important potato pathogen worldwide. Sequence analysis was performed to identify the complete MAPK kinase (MKK) family in potato, and to identify those regulated in the hypersensitive resistance response to <i>Potato virus Y</i> infection. <i>Arabidopsis</i> has 10 MKK family members, of which we identified five in potato and tomato (<i>Solanum lycopersicum</i> L.), and eight in <i>Nicotiana benthamiana</i>. Among these, St<i>MKK6</i> is the most strongly regulated gene in response to <i>Potato virus Y</i>. The salicylic acid treatment revealed that St<i>MKK6</i> is regulated by the hormone that is in agreement with the salicylic acid-regulated domains found in the St<i>MKK6</i> promoter. The involvement of St<i>MKK6</i> in potato defence response was confirmed by localisation studies, where StMKK6 accumulated strongly only in <i>Potato-virus-Y</i>-infected plants, and predominantly in the cell nucleus. Using a yeast two-hybrid method, we identified three StMKK6 targets downstream in the MAPK cascade: StMAPK4_2, StMAPK6 and StMAPK13. These data together provide further insight into the StMKK6 signalling module and its involvement in plant defence.</p></div

Phylogenetic tree of <i>MKK</i> family in <i>A. thaliana</i> and five <i>Solanaceae</i> species.

<p>The species are <i>A. thaliana</i> (At), potato (Sotub), tomato (Solyc), <i>N. benthamiana</i> (Nb), <i>N. attenuata</i> (Na) and <i>N. tabacum</i> (Nt). Genes are grouped into 4 groups: A (green), B (red), C (blue) and D (orange) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104553#pone.0104553-Ichimura1" target="_blank">[4]</a>. Potato genes are marked with dots. The numbers on the nodes are percentages from a bootstrap analysis of 1000 replicates. The scale bar indicates the branch length that corresponds to 0.06 substitutions per site.</p

Yeast two-hybrid assays screening StMKK6 interaction partners.

<p>StMKK6 protein was fused with Gal DNA-binding domain as bait and StMAPK4_1, StMAPK4_2, StMAPK6 and StMAPK13 were used as prey fused with Gal DNA-activation domain. Interaction pairs P53/SV40 large T-antigen and Lam/SV40 large T-antigen were used as positive and negative controls respectively. StMKK6/empty vector pair was used as a control to discard auto-activation of bait protein. Serial dilutions of interaction pairs were plated on DDO media for co-transformation selection. Interactions of bait and prey proteins were examined by assessing growth on several selective media with different levels of restrictiveness i.e. SD/-Leu/-Trp/-His (TDO), SD/-Leu/-Trp/x-a-Gal/Aba (DDO/X/A) and QDO/X/A. Only co-transformed colonies growing on the most restrictive media (QDO/X/A) were considered as positive interaction transformants.</p