[Comparison of DNA Extraction Methods for Processed Foods Containing Soybean or Maize].

Processed foods containing soybean or maize are subject to labeling regulations pertinent to genetically modified (GM) foods in Japan. To confirm the reliability of the labeling procedure of GM foods, the Japanese standard analytical methods (standard methods) using real-time PCR technique have been established. Although certain DNA extraction protocols are stipulated as standard in these methods, the use of other protocols confirmed to be equivalent to the existing ones was permitted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or corn was conducted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or maize was conducted. The silica membrane-based DNA extraction kits, GM quicker 4 and DNeasy Plant Maxi Kit (Maxi Kit), as an existing method were compared. GM quicker 4 was considered to be equivalent to or better than Maxi Kit.

[Investigation of Genetically Modified Maize Imported into Japan in 2021/2022 and the Applicability of Japanese Official Methods].

Given that the number of genetically modified (GM) maize events that have been announced as having undergone safety assessment procedures in Japan is increasing yearly, more information is needed about their actual recent domestic distribution in Japan. In this study, we investigated whether current Japanese official qualitative and quantitative methods (the current official methods) for GM maize can comprehensively target events in domestically distributed maize. For samples with the identity-preserved (IP) handling system and non-IP samples from the United States (US) and non-IP samples from Brazil, we performed event-specific real-time PCR targeting 25 authorized single GM maize events in addition to the current official methods. According to our results, 15 events targeted by the current official methods were detected, but insect-resistance (IR) Event5307 and herbicide-tolerant (HT) DAS40278, not targeted by the current official methods, were detected in the US (one out of 5 lots) and Brazilian (four out of 5 lots) non-IP samples, respectively. Nevertheless, a survey of recent GM maize acreage in recent years has revealed that more than 95% of the acreage in US maize is occupied by HT or IR/HT stacked events, and that more than 95% of the acreage in Brazilian maize is occupied by IR or IR/HT stacked events. Because the current official methods can target all stacked events related to Event5307 and DAS40278, the only undetectable events are the single Event5307 and DAS40278, whose production is estimated to be less than 5% of the total production in the producing country. Therefore, we conclude that the current official methods for the labelling of GM maize should be maintained in view of practicability.

Development and Validation of a New Robust Detection Method for Low-Content DNA Using ΔΔCq-Based Real-Time PCR with Optimized Standard Plasmids as a Control Sample.

Real-time polymerase chain reaction (PCR) is the gold standard for DNA detection in many fields, including food analysis. However, robust detection using a real-time PCR for low-content DNA samples remains challenging. In this study, we developed a robust real-time PCR method for low-content DNA using genetically modified (GM) maize at concentrations near the limit of detection (LOD) as a model. We evaluated the LOD of real-time PCR targeting two common GM maize sequences (P35S and TNOS) using GM maize event MON863 containing a copy of P35S and TNOS. The interlaboratory study revealed that the LOD differed among laboratories partly because DNA input amounts were variable depending on measurements of DNA concentrations. To minimize this variability for low-content DNA samples, we developed ΔΔCq-based real-time PCR. In this study, ΔCq and ΔΔCq are as follows: ΔCq = Cq (P35S or TNOS) - Cq (SSIIb; maize endogenous gene), ΔΔCq = ΔCq (analytical sample) - ΔCq (control sample at concentrations near the LOD). The presence of GM maize was determined based on ΔΔCq values. In addition, we used optimized standard plasmids containing SSIIb, P35S, and TNOS with ΔCq equal to the MON863 genomic DNA (gDNA) at concentrations near the LOD as a control sample. A validation study indicated that at least 0.2% MON863 gDNA could be robustly detected. Using several GM maize certified reference materials, we have demonstrated that this method was practical for detecting low-content GM crops and thus for validating GM food labeling. With appropriate standards, this method would be applicable in many fields, not just food.

Development and Interlaboratory Validation of a Novel Reproducible Qualitative Method for GM Soybeans Using Comparative Cq-Based Analysis for the Revised Non-GMO Labeling System in Japan.

Many countries have implemented the labeling system of genetically modified organisms (GMO). In Japan, the regulatory threshold for non-GMO labeling will be revised and restricted to undetectable by April 2023. The practical criterion for the revised system is based on the limit of detection (LOD). However, determining whether the commingling of GMO levels exceeds the LOD is challenging because GM contents close to the LOD are usually below the limit of quantification. In this study, we developed a qualitative method based on comparative Cq-based analysis targeting cauliflower mosaic virus 35S promoter and GM soybean MON89788 event-specific sequences that could be applicable to the revised non-GMO labeling. ΔCq values between the target and endogenous sequences were calculated, and the ΔΔCq value obtained was used as a criterion to determine analytical samples with GM contents exceeding the threshold. To improve the reproducibility of the method, we used a standard plasmid that yields equivalent and stable ΔCq values comparable with those obtained from LOD samples. The developed method was validated with an interlaboratory study. The new qualitative detection concept would be useful for ensuring robust and reproducible results among laboratories, particularly for detecting low-copy-number DNA samples.