Mari Onishi

Practicable Group Testing Method to Evaluate Weight/Weight GMO Content in Maize Grains

Because of the increasing use of maize hybrids with genetically modified (GM) stacked events, the established and commonly used bulk sample methods for PCR quantification of GM maize in non-GM maize are prone to overestimate the GM organism (GMO) content, compared to the actual weight/weight percentage of GM maize in the grain sample. As an alternative method, we designed and assessed a group testing strategy in which the GMO content is statistically evaluated based on qualitative analyses of multiple small pools, consisting of 20 maize kernels each. This approach enables the GMO content evaluation on a weight/weight basis, irrespective of the presence of stacked-event kernels. To enhance the methods user-friendliness in routine application, we devised an easy-to-use PCR-based qualitative analytical method comprising a sample preparation step in which 20 maize kernels are ground in a lysis buffer and a subsequent PCR assay in which the lysate is directly used as a DNA template. This method was validated in a multilaboratory collaborative trial.

遺伝子組換え(GM)ダイズ新系統MON89788の系統特異的定量検知法の開発および性能指標の評価

A novel real-time PCR-based analytical method was established for the event-specific quantification of a GM soybean event MON89788. The conversion factor (C(f)) which is required to calculate the GMO amount was experimentally determined. The quantitative method was evaluated by a single-laboratory analysis and a blind test in a multi-laboratory trial. The limit of quantitation for the method was estimated to be 0.1% or lower. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were both less than 20%. These results suggest that the established method would be suitable for practical detection and quantification of MON89788.

Development and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12.

A real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event, MON87701. First, a standard plasmid for MON87701 quantification was constructed. The conversion factor (Cf) required to calculate the amount of genetically modified organism (GMO) was experimentally determined for a real-time PCR instrument. The determined Cf for the real-time PCR instrument was 1.24. For the evaluation of the developed method, a blind test was carried out in an inter-laboratory trial. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined biases and the RSDr values were less than 30 and 13%, respectively, at all evaluated concentrations. The limit of quantitation of the method was 0.5%, and the developed method would thus be applicable for practical analyses for the detection and quantification of MON87701.

Development and evaluation of event-specific quantitative pcr method for genetically modified soybean MON89788

Abstract There is widespread use of genetically modified (GM) crops for food and feed in many countries. Several countries have required labelling of all foods containing or derived from authorized GMOs to keep consumers their freedom of choice. To establish a labelling system, it is necessary to define an effective threshold level and to develop validated quantitative methods for the unintentional commingling of GMOs. The thresholds were set at 0.9, 3, and 5% in the European Union (EU), Korea, and Japan, respectively. In Japan, quantitative methods using real-time PCR of several GM maize events and Roundup Ready soy have already been developed and adopted as Japanese standard analytical methods. One of the features of these methods is the utilization of plasmid DNAs containing both recombinant DNA (r-DNA) and endogenous sequences as calibrators. In this study, we developed a real-time PCR-based quantitative method for the event-specific quantification of GM soybean event: MON89788 which is a newly approved glyphosate-tolerant soybean. The conversion factor (Cf) is defined as the experimentally determined ratios of copy number of r-DNA to endogenous taxon-specific sequence in the extracted DNA from each genuine GM seed. We determined Cf value for MON89788 and evaluated the quantitative method by a blind test in a multilaboratory trial. The limit of quantitation was estimated to be 0.1%. The trueness and precision were evaluated as bias and reproducibility of relative standard deviation (RSDR), and the determined bias and RSDR values were both less than 20%. All of the obtained results suggest that the developed method is suitable for the practical l f h d i d ifi i f MON89788 ana yses or t e etect on an quant cat on o .