| The reference material is the "weight" of analysis and testing,which provides an important guarantee for the comparability,effectiveness and traceability of the test results.In the field of transgenic detection,reference materials are very important for the accurate analysis of genetically modified ingredients and their contents in genetically modified foods and feeds,while the research on reference materials is in its infancy.The determination,preparation and uncertainty evaluation of measurement need to be improved and perfected.This paper took the genetically modified soybean MON87751 developed by Monsanto as the research object,focusing on the existing problems in the field of reference materials research,carried out the following three aspects of research:1.An absolute quantitative method for double droplet digital PCR(ddPCR)of reference materials was established.At present,MON87751 soybean was mainly measured by real-time fluorescence quantitative PCR(qPCR)in the world,but there was no report of absolute quantitative analysis by ddPCR,and there was no detection method of this transformant in China.In order to solve this problem,based on the absolute quantification ddPCR platform for nucleic acid,a dual ddPCR method for MON87751 was established.The research contents covered the specificity,repeatability,linear kinetic range,detection limit and quantitative limit of ddPCR,so as to determine the reaction system and reaction conditions.Ten groups of 10% MON87751 soybean samples were analyzed,and the results showed that the relative standard deviation(RSD)of repeatability was between0.10% and 0.71%.When the concentration of template DNA was as low as 4copies,the RSD of the measured value between replicates was 16.95%,which was less than25%(required by the European Union).The quantitative limit of ddPCR was 4copies.2.The reference materials of three concentration gradients of MON87751 soybean were prepared by weight proportion method.In this study,genetically modified soybean MON87751 and its receptor material MON87751 were identified as raw materials to meet the needs of matrix reference materials.The water content of transgenic soybean MON87751 transformant powder was 4.53%,and the receptor water content was 4.40%,which was lower than the prescribed 10%.The ratio of DNA extraction efficiency of transformants to receptors was 1.033.The grinding time of 5 min was verified by laser particle size analyzer,and the transgenic soybean MON87751 transformants and receptors were ground intopowder.MON87751 matrix reference material was prepared according to the mass fraction,and the matrix reference material of transgenic soybean MON87751 was99.8 g/kg,9.9 g/kg and 1.0 g/kg,and measured by ddPCR method,the results were10.04%,1.00%,0.11%.By statistical comparison,there was no significant difference between the results of the two methods,and the consistency was good.As a result,the value of the quantity could be traced back to the SI unit by the weight method and transferred to the next laboratory by the ddPCR method.3.The uncertainty model of MON87751 soybean matrix reference material was established.The mass fraction of the ratio of gravimetric method was used as the standard value of this batch of reference materials,and the uncertainty was evaluated.The results were(99.8±0.034)g/kg,(9.9±0.072)g/kg,(1.0±0.091)g/kg.The intra-bottle and inter-bottle homogeneity of matrix reference materials were analyzed,and the results showed that the intra-bottle and inter-bottle homogeneity of the three gradient reference materials were good.The stability test showed that the matrix reference material could be transported at room temperature(25 ℃)for 14 days and stored stably at-20 ℃ for 12 months.The characteristic values and uncertainty of three gradient matrix reference materials were tested and analyzed by ddPCR,and the results were expressed as(10.04±0.034)%,(1.00±0.071)%,(0.11±0.090)%.The genetically modified soybean MON87751 matrix reference material was packed separately with 1g per bottle,and the minimum sampling amount was 100 mg.In summary,through the study of three key technologies of reference materials,the quantitative value,metrological traceability and measurement uncertainty of genetically modified soybean MON87751 matrix were determined,which could be used in the qualitative and quantitative detection of MON87751,as well as method evaluation and laboratory quality control and other fields.At the same time,it provided a technical reference for the development of standard reference materials for the detection of genetically modified products and nucleic acid reference materials in related fields. |
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