Development and Performance Evaluation of a New Test Kit for Quantifying the Degree of DNA Fragmentation.

BACKGROUND: PCR-based genetic testing of agricultural products and foods is widely used for detecting various analytical targets such as genetically modified organisms and food allergens. However, it is difficult to obtain accurate genetic testing results from processed foods because DNA is fragmented by heat and pressure during food processing. Thus, we previously developed an analytical method to quantitatively evaluate the degree of DNA fragmentation for the purpose of QC of genetic testing for processed foods. OBJECTIVE: Our previous analytical method requires four PCR primer sets, resulting in high reagent costs and heavy analytical workloads. Therefore, we attempted to develop an easy-to-use test kit for quantifying the degree of DNA fragmentation and to evaluate its analytical performance. METHODS: To simplify the analysis procedure, we used only two primer sets. In addition, no-fragmentation control templates were prepared to obtain stable measurement results. The precision of the simplified analysis was evaluated through blind tests between laboratories. RESULTS: It was confirmed that plant species and extracted DNA concentrations had little effect on analysis with the newly developed test kit. In addition, the analytical values indicating the degree of DNA fragmentation exhibited small variability between laboratories. CONCLUSION: We confirmed the high practicality of the developed test kit. Because DNA fragmentation in cells is a universal phenomenon, we anticipate that the test kit will be used not only for QC of genetic testing but also for food testing, medical diagnostics, and other applications in a range of fields. HIGHLIGHTS: The newly developed test kit enables quantitative evaluation of the degree of DNA fragmentation in a simple manner.

Simple, Precise, and Less Biased GMO Quantification by Multiplexed Genetic Element-Specific Digital PCR.

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.