Background: To provide the consumer with choices of genetically modified organisms (GMO) or non-GMO, official food labeling systems were established in many countries. Because the threshold GMO content values were set to distinguish between “non-GMO” and “GMO” designations, GMO content quantification methods are required for ensuring the appropriateness of labeling. Objective: As the number of GMOs is continuously increasing around the world, we set out to develop a low-cost, simple and less biased analytical strategy to cover all necessary detection targets. Methods: Digital PCR methods are advantageous compared to the conventional quantitative real-time PCR methods. We developed a digital PCR-based GMO quantification method to evaluate the GMO content in maize grains. To minimize the analytical workload, we adopted multiplex digital PCR targeting the 35S promoter and the nopaline synthase terminator, which are genetic elements commonly introduced in many GMOs. Results: Our method is significantly simpler and more precise than the conventional real-time PCR-based methods. Additionally, we found that this method enables quantification of the copy number of GMO DNA without double counting multiple elements (35S promoter and nopaline synthase terminator) tandemly placed in a recombinant DNA construct. Conclusion: This is the first report on the development of a genetically modified maize quantification method using a multiplexed genetic element-specific digital PCR method. The tandem effect we report here is quite useful for reducing the bias in the analytical results. Highlights: Multiplexed genetic element-specific digital PCR can simplify weight-based GMO quantification and thus should prove useful in light of the continuous increase in the number of GM events.

The continuous increase in the global population has led to a rise in the demand for protein. However, conventional meat production has adverse environmental effects. Thus, developing alternative ways to produce edible protein with high efficiency and low cost is necessary. In this study, a novel protein production process was developed based on traditional koji fermentation, i.e., solid-state Aspergillus oryzae cultivation on grains. By supplementing nitrogen sources into rice and maize grains, the protein content of fermented products reached to 12.3% and 13.2% and the amount of total protein was increased to 2.32-fold and 1.58-fold, respectively. In addition, koji fermentation enhanced the content of lysine, which is the limiting amino acid for humans and livestock. Nitrogen-supplemented koji fermentation is a practical technology for the quick protein enrichment of cereal grains without the need for advanced facilities, indicating its great application potential in alleviating the anticipated global shortage of protein-rich foods.