种子粉基体黄瓜绿斑驳花叶病毒标准样品研制及应用

Preparation of Cucumber Green Mottle Mosaic Virus in Seed Powder Matrix Reference Material

基于国内黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus, CGMMV)标准样品缺失的问题,以自然感染CGMMV的西葫芦种子批为原料,经特异性鉴定确认后,制备了西葫芦种子粉基体CGMMV标准样品,采用双抗体夹心酶联免疫吸附法和实时荧光定量RT-PCR法对其进行确证。结果表明所制备的标准样品性能稳定,经国内8家实验室对样品进行协同定值后,再经Shapiro-Wilk检验均符合正态分布。此外,采用Grubb’s法检验进一步证实该种子粉基体标准样品中含有CGMMV。以上结果表明,该基体标准样品达到了国家标准样品的技术要求,或可用于实验室质量控制及能力验证、检测方法验证等活动。

Seed powder matrix reference materials for cucumber green mottle mosaic virus were developed in this study to resolve the problem of lack of qualitative standard sample.Seed batches of Cucurbita pepo L.naturally infected with CGMMV were used as raw materials.After the specificity identification and confirmation,CGMMV reference materials of Cucurbita pepo L.seed powder matrix were prepared,which has been confirmed by double antibody sandwich enzyme-linked immunosorbent(DAS-ELISA)assay and real-time fluorescence quantitative RTPCR(RT-qPCR)method.The results demonstrated that the reference materials were stable.After the samples were certified by using collaborative characterization program with eight sophisticated participating laboratories,Shapiro Wilk test showed that the standard sample conforms to Normal distribution.Seed power matrix reference materials were certified contained CGMMV by Grubb s method.Based on above results,the matrix reference materials in accordance with the requirements of the national reference material,it can be used for laboratory quality control,capability verification,testing method verification,and other activities.