Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events

• Published in: Analytical and bioanalytical chemistry 2015-04, Vol.407 (10), p.2877-2886
• Main Authors: Pi, Liqun, Li, Xiang, Cao, Yiwei, Wang, Canhua, Pan, Liangwen, Yang, Litao
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Biochemistry; Calibration; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Corn; Crops; DNA sequencing; Food; Food Science; Fractionation; Gene sequencing; Genetic aspects; Genetically altered foods; Genetically modified organisms; Glycine max - genetics; Labeling; Laboratory Medicine; Lectins; Limit of Detection; Mass spectrometry; Monitoring/Environmental Analysis; Nucleotide sequencing; Plant Lectins; Plants, Genetically Modified - genetics; Plasmids; Plasmids - genetics; Quarantine; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Reference materials; Regression analysis; Reproducibility of Results; Reproduction; Research Paper; Rice; Seeds; Sensitivity and Specificity; Soybean; Soybean Proteins; Soybeans; Standard deviation
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-015-8517-5

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. Graphical Abstract One multi-targets plasmid, pSOY, that can be used as calibrator in the detection of the five GM soybean Events