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.
Highlights
In many aspects of basic research, diagnostic tests, and commercial processes, the advent of modern analytical technologies has provided the ability to detect and quantify nucleic acid targets with unprecedented sensitivity and specificity
Apart from the mastermix and settings that are specific to the QX100 droplet system, primers and probe nucleotide sequences and concentrations, DNA concentration, and polymerase chain reaction (PCR) thermo-profile were kept identical to those in the quantitative PCR (qPCR) assays
DdPCR can readily be set as a duplex application Because genetically modified organisms (GMOs) content is calculated based on the ratio of transgene/endogene quantities, it would be more practical to perform endogene–transgene duplex reactions to reduce costs
Summary
In many aspects of basic research, diagnostic tests, and commercial processes, the advent of modern analytical technologies has provided the ability to detect and quantify nucleic acid targets with unprecedented sensitivity and specificity. Its use for target quantification can be seriously limited by a significant bias when the target is present at low concentrations in a background of high numbers of non-target nucleic acids in the sample [4,5,6,7]. Another important limitation is its sensitivity to the frequent presence of inhibitors coextracted with nucleic acid from complex matrices [8]. Numerous countries have implemented regulations requiring the labeling of products containing GMOs, or materials derived from GMOs, above certain thresholds, emphasizing the requirement for quantification of GMO content [9]
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