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. 2013 May 2;8(5):e62583.
doi: 10.1371/journal.pone.0062583. Print 2013.

Quantitative analysis of food and feed samples with droplet digital PCR

Affiliations

Quantitative analysis of food and feed samples with droplet digital PCR

Dany Morisset et al. PLoS One. .

Abstract

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 hmg 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Dynamic range of the ddPCR duplex assay.
Five replicates for each data point. Error bars represent the standard deviation between the five replicates at each target concentration.
Figure 2
Figure 2. Repeatability results of the ddPCR duplex assay.
MON810 content measured by ddPCR in five series of seven replicates. The aggregate represents the sum of the five series. The target certified MON810 content (0.77%) is indicated by a dotted line. Acceptance criterion for repeatability is ±25% of the target content (from 0.58% to 0.96%) represented by the dashed lines. Error bars represent the standard deviation between the replicates for each series or in the aggregate.
Figure 3
Figure 3. Precision of the duplex ddPCR assay as a function of the target concentration.
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).

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