Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR

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.

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

“…While the ddPCR assay was lower in sensitivity, other criteria required for operation are comparable to or even surpass those of the qPCR assay. ddPCR is known to provide several advantages over qPCR: it is less susceptible to PCR inhibition and high background DNA levels; it gives absolute quantification without relying on an external standard reference; and it is more precise than qPCR (20)(21)(22). Our findings are in agreement with those previous observations showing that the ddPCR method was indeed higher in precision for either plasmid standard or environmental sample analysis.…”

“…The intrinsic nature of ddPCR allows detection of rare nucleic acids in the presence of a high abundance of standard sequences (15). The applications of this technology have mainly been focused on clinical research and diagnosis, genetic modification, food product screening, and viral surveillance (17)(18)(19)(20)(21)(22)(23), while the potential of using ddPCR in environmental studies has yet to be explored.…”

Next Generation Digital PCR Measurement of Hepatitis B Virus Copy Number in Formalin-Fixed Paraffin-Embedded Hepatocellular Carcinoma Tissue

“…This method relies on limiting dilutions of the PCR volume and Poisson statistics (9,14 ). After the dilutions, the PCR mix is distributed into fractions, and each reaction is independently interrogated for the copy number of the target nucleic acids at a single-molecule sensitivity (15 ).…”

New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

“…the number of dyes that can currently be detected, the occurrence of false positives when the targets to be multiplexed become too numerous and/ or may interact and the loss of sensitivity. Furthermore, high-throughput technologies, as, for instance, NASBA implemented microarray analysis (NAIMA) [23,24], microdroplet PCR implemented capillary gel electrophoresis (MPIC) [25], multiplex ligation detection methods [26] (and references therein), a combined microchip-PCR and microarray system (MACRO) [27], digital PCR [28,29] and next-generation sequencing (NGS) [30][31][32], have been developed and their possible use in GMO detection was demonstrated. These methods are, however, still too costly and/or cumbersome for routine use and often require expensive equipment and/or specialised data analysis tools and staff.…”