乙型肝炎病毒DNA二级标准物质的研制

doi:10.3969/j.issn.1673-8640.2016.08.016

摘要/Abstract

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=1.052 4,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。

关键词: 标准物质, 肝炎病毒, 乙型, DNA

Abstract:

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 1.00×10⁶IU/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）×10⁶IU/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.

Key words: Reference materials, Hepatitis B virus, DNA

中图分类号:

R446.62

蒋玲丽, 王华梁, 王雪亮, 鲍芸, 肖艳群. 乙型肝炎病毒DNA二级标准物质的研制[J]. 检验医学, 2016, 31(8): 703-708.

JIANG Lingli, WANG Hualiang, WANG Xueliang, BAO Yun, XIAO Yanqun. Establishment of a national grade Ⅱ reference material for HBV DNA[J]. Laboratory Medicine, 2016, 31(8): 703-708.

图/表 7

参考文献 10

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检测次数	-20 ℃	开瓶7 d	冻融15次
1	6.31	6.25	6.39
2	6.34	6.35	6.33
3	6.29	6.39	6.39
4	6.43	6.32	6.43
5	6.34	6.30	6.43
6	6.29	6.31	6.29
x	6.33	6.32	6.38
t值		-0.35	0.46
P值		>0.05	>0.05

检测次数	-20 ℃	开瓶7 d	冻融15次
1	6.31	6.25	6.39
2	6.34	6.35	6.33
3	6.29	6.39	6.39
4	6.43	6.32	6.43
5	6.34	6.30	6.43
6	6.29	6.31	6.29
x	6.33	6.32	6.38
t值		-0.35	0.46
P值		>0.05	>0.05

检测次数	-20 ℃	崇明中心医院	青浦中心医院
1	6.29	6.44	6.28
2	6.39	6.42	6.33
3	6.38	6.38	6.39
4	6.40	6.36	6.36
5	6.28	6.46	6.32
6	6.43	6.39	6.36
x	6.37	6.41	6.34
t值		1.53	0.75
P值		>0.05	>0.05

检测次数	-20 ℃	崇明中心医院	青浦中心医院
1	6.29	6.44	6.28
2	6.39	6.42	6.33
3	6.38	6.38	6.39
4	6.40	6.36	6.36
5	6.28	6.46	6.32
6	6.43	6.39	6.36
x	6.37	6.41	6.34
t值		1.53	0.75
P值		>0.05	>0.05

不确定度来源	x（IU/mL）	s（IU/mL）	u（%）
u_均匀性rel	1.47×10⁶	9.43×10⁴	6.41
u_稳定性rel	1.70×10⁶	2.23×10⁵	13.11
u_定值rel	2.5×10⁶	1.07×10⁵	10.85
u_crel			18.20
U_rel			36.00