4种阪崎肠杆菌检测质粒DNA标准物质的研制  

Development of Four Plasmid DNA Reference Materials for Cronobacter spp. Detection

作　　者：许丽[1] 梁文[1] 李妍[1] 任淑贞[1] 刘刚[1] 

机构地区：[1]上海市计量测试技术研究院化学与电离辐射计量技术研究所,上海201203

出　　处：《实验室研究与探索》2016年第3期4-8,共5页Research and Exploration In Laboratory

基　　金：国家自然科学青年基金项目(21305091);国家质检总局科技计划项目(2012QK273);国家质检总局科技计划项目(2014QK141);国家质检公益性行业科研专项(201310016);上海市自然科学基金青年基金(12ZR1448300);上海市科委科研计划项目(13QB1402900)

摘　　要：实时荧光定量(PCR)方法已成为阪崎肠杆菌的可靠检测方法。为了保证该方法检测结果的准确性、可靠性和可比性,研制了4种质粒DNA标准物质,其分别含有一种阪崎肠杆菌PCR检测靶基因(MMS,ITS,16S r DNA,omp A)。采用紫外分光光度法(UV)和高分辨电感耦合等离子体质谱法(HR-ICP-MS)对标准物质进行定值,对该标准物质的均匀性、稳定性进行了评估,并对定值不确定度进行了评定。结果显示,4种质粒DNA标准物质量值可靠,均匀性和稳定性良好,可以在-20℃下保存1 a以上。4种质粒DNA标准物质的最终定值结果为(52.3±1.8)、(62.4±2.1)、(75.1±2.1)和(85.7±2.6)ng/μL(k=2)。对质粒DNA标准物质与阪崎肠杆菌基因组DNA的可替代性进行了研究,证明研制的质粒DNA标准物质可以替代阪崎肠杆菌基因组DNA对阪崎肠杆菌进行检测,满足阪崎肠杆菌检测实验室质量控制和方法验证的需求。Real-time quantitative PCR has become a reliable method to detect Cronobacter spp. Four Cronobacter spp.related plasmid DNA reference materials（ RMs） were designed to contain respectively one of four main PCR target genes（MMS,ITS,16 S r DNA,omp A）. RMs are commonly used in PCR-based detection of Cronobacter spp. All RMs were analyzed by both high resolution inductively coupled plasma mass spectrometry（ HR-ICP-MS） and ultraviolet spectrophotometry（ UV）. Nine different laboratories participated in the quantification of the RMs,and the uncertainties of these quantification results were evaluated. The analysis results demonstrated that the four reference materials were homogeneous and stable for at least one year under- 20℃ storage. The certified value and expanded uncertainty of the reference materials were as follows：（ 52. 3 ± 1. 8）,（ 62. 4 ± 2. 1）,（ 75. 1 ± 2. 1） and（ 85. 7 ± 2. 6） ng / μL,respectively（ k = 2）. The perfect substitutability of Cronobacter spp. genomic DNA shows that the four RMs can replace the Cronobacter spp. genomic DNA in quality control of real-time PCR detection. The development of the four RMs has provided scientific basis for the traceability of value of Cronobacter spp. real-time PCR detection.

关 键 词：质粒DNA 标准物质 实时荧光 阪崎肠杆菌 

分 类 号：Q819[生物学—生物工程]