乙型肝炎病毒 DNA 二级标准物质的研制

摘要

目的：研制乙型肝炎病毒（HBV）DNA血清二级国家标准物质。方法用HBV阴性血清将阳性血清稀释为1.00×106 IU/mL，以1.5 mL/支分装。评价采用Roche COBAS AmpliPrep/COBAS TaqMan 48检测系统。制备后评价基质互通性；抽取制备物30支，每支检测2次，评价均匀性；检测室温14 d、37℃7 d、45℃3 d、2～8℃6个月和-20℃12个月的稳定性；评价反复冻融15次、开瓶后放置7 d和模拟运输后的稳定性。定值通过溯源至国家一级标准物质（GBW09150），不确定度包括不均匀性引入的不确定度、长期稳定性引入的不确定度和定值时引入的不确定度。结果基质互通性实验表明制备物测定的均值在临床新鲜样本95%可信区间内；均匀性实验表明批间不精密度为2.04%，批内不精密度为1.98%（F=1.0524， P>0.05）；室温14 d、37℃7 d、45℃3 d、2～8℃6个月和-20℃12个月的检测结果与第0天/月比较，采用直线拟合法分析，差异无统计学意义（P>0.05）；反复冻融15次、开瓶后放置7 d和模拟运输（至2家实验室）的检测结果与对照组（-20℃）比较，差异无统计学意义（t=0.46、-0.35、1.53、0.75，P>0.05）。制备物定值为（2.5±0.9）×106 IU/mL。结论制备物达到了国家二级标准物质的要求，可用作核酸扩增检测的HBV DNA标准物质。%Objective To establish a national grade Ⅱ reference material for hepatitis B virus(HBV) DNA. Methods The HBV positive serum was diluted with HBV negative serum. The content was 1.00×106 IU/mL, packed to 1.5 mL each bottle. Roche COBAS AmpliPrep/COBAS TaqMan 48 testing system was used. Matrix interoperability was evaluated. For homogeneity evaluation,30 bottles were randomly selected,and each bottle was determined for 2 times. The prepared materials were placed at room temperature for 14 d,37 ℃ for 7 d,45 ℃ for 3 d,2-8 ℃ for 6 months and -20 ℃ for 12 months,and the stabilities were evaluated. The stabilities of the prepared materials which were repeated freezing and thawing for 15 times,7 d after the opening of bottles and simulated transportation to 2 laboratories were evaluated. The quantity of prepared materials was traced to the national standard material(GBW09150). The uncertainties included the introduction of heterogeneity,the long-term stability and the quantity. Results Matrix interoperability test showed that the prepared materials' mean value was within the 95%confidence interval of clinical fresh samples. The between-run imprecision was 2.04%,and the within-run imprecision was 1.98%(F=1.052 4,P>0.05). The stability test indicated the prepared materials were stable at room temperature for 14 d,37 ℃ for 7 d,45 ℃ for 3 d,2-8 ℃ for 6 months and -20 ℃ for 12 months,and the slope rates had no statistical significance compared with the 0th day/month by linear fitting test(P>0.05). The stability was observed in prepared materials which were repeated freezing and thawing for 15 times,7 d after the opening of bottles and simulated transportation to 2 laboratories with no statistical significance compared with prepared materials which stored at -20 ℃(t=0.46,-0.35,1.53 and 0.75,P>0.05). The quantity was(2.5±0.9)×106 IU/mL. Conclusions The prepared materials have reached the requirements of national grade Ⅱ reference material,which can be used as a reference material for nucleic acid amplification for HBV DNA.