title: Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events.
abstract: Reference materials are important in accurate analysis of genetically modified organism (GMO) contents in food/feeds, and development of novel reference plasmid is a new trend in the research of GMO reference materials. Herein, we constructed a novel multi-targeting plasmid, pSOY, which contained seven event-specific sequences of five GM soybeans (MON89788-5', A2704-12-3', A5547-127-3', DP356043-5', DP305423-3', A2704-12-5', and A5547-127-5') and sequence of soybean endogenous reference gene Lectin. We evaluated the specificity, limit of detection and quantification, and applicability of pSOY in both qualitative and quantitative PCR analyses. The limit of detection (LOD) was as low as 20 copies in qualitative PCR, and the limit of quantification (LOQ) in quantitative PCR was 10 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and Lectin assays were higher than 90%, and the squared regression coefficients (R(2)) were more than 0.999. The quantification bias varied from 0.21% to 19.29%, and the relative standard deviations were from 1.08% to 9.84% in simulated samples analysis. All the results demonstrated that the developed multi-targeting plasmid, pSOY, was a credible substitute of matrix reference materials, and could be used as a reliable reference calibrator in the identification and quantification of multiple GM soybean events.
authors: Collaborative Innovation Center for Biosafety of GMOs, National Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors: Liqun
authors: L
authors: Pi
authors: Xiang
authors: X
authors: Li
authors: Yiwei
authors: Y
authors: Cao
authors: Canhua
authors: C
authors: Wang
authors: Liangwen
authors: L
authors: Pan
authors: Litao
authors: L
authors: Yang
mesh_terms: D002138:Calibration; D057230:Limit of Detection; D037121:Plant Lectins; D030821:Plants, Genetically Modified / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D030262:Soybean Proteins; D013025:Glycine max / Q000235:genetics*; D010835:Phytohemagglutinins
chemical_list: D037121:Plant Lectins; D030262:Soybean Proteins; C028824:soybean lectin; D010835:Phytohemagglutinins