Development Of Seven Plasmids Reference Materials For Detection Of Genetically Modified Rice

Reference materials are widely used in genetically modified crops analysis and development of GM reference materials is vital to improve the GMO detection methods and equipments.In this study,we construct four single-target plasmid reference mulecules and a multi-target plasmid reference molecule for genetically modified rice detection.Relevant applicability assessment,homogeneity evaluation,and stability test were performed using these molecules as calibrators,the droplet digital PCR(dd PCR)platform was utilized to obtain the certified values.First part focused on developing and evaluating four novel single-target plasmid reference molecules for detection of GM rice PA110-15,G6H1,G281 and 4114-7-2.These molecules all contained corresponding event-specific fragment,a 279 bp SPS fragment and a2463bp rice genomic DNA fragment.The results showed that the PCR efficiencies were 96%-98%and the squared regression coefficients(R~2)were 0.9994-1.0000 in quantitative real-time PCR analysis.The relative standard deviations of reproducibility varied from 0.09%to 2.21%and the relative standard deviations of reproducibility varied from 0.10%to 1.25%.The limits of detection(LODs)were 5 copies/μL and the limits of quantitation(LOQs)were about 10 copies/μL.The result of homogeneity and stability assessment indicated the three plasmid reference materials(pPA110-15,pG6H1 and pG281)have good homogeneity and could be stored at least half a year stably.The certified values from ddPCR technology were close to the theoretical value of 1(copy/copy).The quantification bias of blind samples analysis were 0.60%-5.97%,the relative standard deviations were 0.69%-9.53%.The above mentioned values were within the standard range of ENGL guideline.All of these indicated that our developed three single-target plasmid reference materials are reliable and suitable for the identification and quantification of the GM rice events.The second part refered to development and application of a multi-target plasmid reference molecule which can be used to detect six GM rice events(TT51,KF2,KMD,KF6,KF8 and M12).Apart from a SPS fragment(279bp)and a 2463bp rice genomic DNA fragment,six event-specific sequences were constructed into the pRICE-6 molecule.We evaluated the specificity and sensitivity in qualitative PCR system and analysis the application in quantitative PCR assay.According to the results,the sensitivity of event-specific fragments and SPS fragment were all 20copies/μL.The five quantitative standard curves(TT51、KF6、KF8、M12and SPS)were constructed using pRICE-6 as calibrator,and all of them had high PCR efficiency(94%-99%)and good linearity(0.9993-0.9999).The quantification bias ranged from 0.19%to 16.61%,and the relative standard deviations ranged from 1.16%to 8.39%in simulated samples analysis.All of these results were complying with the ENGL performance criteria requirements,and indicating that the developed multi-target plasmid molecule pRICE-6 can be used as a reliable reference material to analyse GM rice TT51、KF6、KF8 and M12 with high throughput and low cost.