基于核酸检测的虾过敏源标准物质研究

Study on reference materials of shrimp allergen based on nucleic acid detection

选择最常食用的海产对虾作为虾过敏源标准物质的候选物,提取并纯化其基因组DNA,采用序列测定法对设定的目标基因成分进行定性鉴定。用数字PCR技术精确确定海虾16S基因、虾真核生物18S基因、胡萝卜真核生物18S基因的拷贝数,以胡萝卜基因组DNA为本底,重量法配置虾16S基因拷贝数/真核生物18S基因组拷贝数比值为1%。评定虾16S基因拷贝数/真核生物18S基因拷贝数比值的定值不确定度,扩展不确定度为0.09%。并利用数字PCR仪对配置的标准物质进行特性量值验证,结果一致性良好。可为基于核酸检测的食物过敏源标准物质的研制提供方法参考。

The marine shrimp was selected as a candidate for shrimp allergen reference materials,and its genomic DNA was extracted and purified.The target gene components were identified qualitatively by sequencing.The copies of shrimp 16S gene,shrimp and carrot eukaryotic18S gene were accurately determined by digital PCR.Based on the genomic DNA of shrimp 16S gene,shrimp and carrot eukaryotic 18S gene,the ratio for copies of 16S gene/18S gene of eukaryotic certified by gravimetric method was 1%.The uncertainty of DNA reference materials for the ratio for copies of 16S gene/18S gene of eukaryotic was evaluated,and the expanded uncertainty was 0.09%.Digital PCR was used to validate the characteristic values of the standard substances,and the results were in good agreement,which provided a reference for the development of food allergens reference substances based on nucleic acid detection in China.