对转基因玉米T25转化体特异性荧光定量PCR方法的关键参数进行了方法验证,选择了7家实验室采用经过验证的方法对3个水平的转基因玉米T25基体标准物质进行协同实验研究.通过比较分析各参与实验室的实验条件、标准曲线的参数,进一步证明该方法室间重复性好.经统计学分析各参与实验室的数据等精度,因此计算各实验室的总平均值做为标准物质的标准值.3个水平的基体标准物质的标准值和扩展不确定度(k=2)分别为1.17％±0.22％、2.17％±0.38％和9.81％±2.30％.

Enterobacter sakazakii is a foodborne pathogen.Currently,there are a growing number of molecular detection techniques for the detection of Enterobacter sakazakii in order to replace the traditional detection techniques.To develop the corresponding nucleic acid reference materials is imperative for the molecular detection technology.Nucleic acid extraction and purification is a key link of development in nucleic acid reference materials.With characteristic of rapid,efficient,high-quality,low toxicity and low cost has become an important target of nucleic acid extraction.In this paper,we compared some existing methods for extracting DNA,especially focusing on three kinds of DNA extraction kid methods for extracting genome DNA from microbe,and obtained a satisfactory method for extracting genome DNA form Enterobacter sakazakii.Additionally,we used specially amplified PCR detection for extracted genome DNA,and obtained a clearer band,therefore laying the foundation for the development in nucleic acid reference materials.

The candidate material properties and its effect on the characteristic value of gene modified reference materials are studied from the water content, particle size and genetic purity. The reference materials of GM rice seed powder are developed with gravimetric method, and the detection method of homogeneity by combining the nano-Ag marker with inductively coupled plasma mass-spectrometric technique is established. The reference materials of GM rice seed powder are developed, the homogeneity and stability are tested, the minimum sample size are determined, the consistency between the preparation values with the gravimetric method and determination values with fluorescence quantitative PCR is analyzed. The GM rice reference materials can be used in laboratory quality control and quantitative determination of gene modified components in rice.

The reference material of adenosine-5’-phosphoric acid was characterized by mass spectrometry and infrared spectroscopy. The purity was determined by high performance liquid chromatography (HPLC), the separation and detection conditions were studied.The resolution of HPLC was influenced by the organic solvent, acidity, inorganic salts and their concentration. The homogeneity and stability of the reference material were described. The value of the purity was co-certified by a net work of Labs using HPLC. The purity and the expanded combined uncertainty were 99.61% and 0.13%(k=2) respectively.

The reference material,flavor nucleotides,has been prepared through the recrystallization and purification of commercially available disodium inosinate,which was characterized by Mass spectrometry and infrared spectroscopy.The purity was determined by high performance liquid chromatography (HPLC) and the separation and detection conditions were studied.It was found that the organic solvent can improve the retention time of disodium inosinate and the acidity and ionic concentration can improve the separating size of chromatogram.The homogeneity and stability of the reference material were also described.Its purity and the expanded combined uncertainty were 99.75% and 0.32%(k=2) respectively.

The mass of oligonucleotides was quantitated by liquid chromatography-isotope dilution mass spectrometry.The uncertainty was calculated.This could provide information for the further quantitative measurement and utilization of DNA.