BCR-ABL1候选参考物质的研制及测量不确定度评定

doi:10.3969/j.issn.1673-8640.2019.08.017

摘要/Abstract

摘要：

目的构建慢性粒细胞白血病（CML）融合基因断裂点簇集区-埃布尔森小鼠白血病病毒癌基因1（BCR-ABL1）候选参考物质。方法采用分子克隆技术构建含有BCR-ABL1基因的重组质粒,测序验证后采用紫外分光光度法初步测定浓度,并采用数字聚合酶链反应（dPCR）进行浓度定值,实时荧光定量聚合酶链反应（RQ-PCR）进行均匀性和稳定性研究,最终应用《测量不确定度表示指南》评定测量不确定度。结果质粒候选参考物质拷贝数浓度为（2.50±0.35）×10⁶ 拷贝/μL,有较好的均匀性和稳定性。结论建立了制备BCR-ABL1候选参考物质的方法,构建了均匀稳定的BCR-ABL1候选参考物质。

关键词: 断裂点簇集区-埃布尔森小鼠白血病病毒癌基因1, 参考物质, 测量不确定度, 数字聚合酶链反应, 紫外分光光度法

Abstract:

Objective To develop the breakpoint cluster region-abelson murine leukemia viral oncogene homolg 1 （BCR-ABL1） fusion gene candidate reference material for chronic myeloid leukemia（CML）. Methods A plasmid containing the BCR-ABL1 fusion gene was constructed by molecular cloning technology,and its concentration was quantified by ultraviolet spectrophotometry and digital polymerase chain reaction（PCR） following sequence validation. The homogeneity and stability were evaluated by real-time quantitative PCR（RQ-PCR）. The uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement. Results The copy concentration of plasmid candidate reference material was（2.50±0.35）×10⁶ copies/μL. The homogeneity and stability of plasmid were good. Conclusions The method for constructing BCR-ABL1 candidate reference material and a homogeneous and stable BCR-ABL1 candidate reference material have been established.

Key words: Breakpoint cluster region-abelson murine leukemia viral oncogene homolg 1, Reference material, Measurement uncertainty, Digital polymerase chain reaction, Spectrophotometry

中图分类号:

R446.1

于书平, 袁丹丹, 崔明, 景蓉蓉, 王惠民. BCR-ABL1候选参考物质的研制及测量不确定度评定[J]. 检验医学, 2019, 34(8): 752-757.

YU Shuping, YUAN Dandan, CUI Ming, JING Rongrong, WANG Huimin. Development of BCR-ABL1 candidate reference material and evaluation of measurement uncertainty[J]. Laboratory Medicine, 2019, 34(8): 752-757.

图/表 8

参考文献 12

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基因名称	引物序列（5'~3'）	预期扩增产物长度（bp）
BCR-ABL1 F1	TGTCGGAGCAGGAGTCAC	1 055
BCR-ABL1 R1	CACCGTCAGGCTGTATTT
BCR-ABL1 F2	GGGCTCTATGGGTTTCTGAATG	77
BCR-ABL1 R2	CGCTGAAGGGCTTTTGAACTC
BCR-ABL1 P	FAM-ATCGTCCACTCAGCCAC-MGB

基因名称	引物序列（5'~3'）	预期扩增产物长度（bp）
BCR-ABL1 F1	TGTCGGAGCAGGAGTCAC	1 055
BCR-ABL1 R1	CACCGTCAGGCTGTATTT
BCR-ABL1 F2	GGGCTCTATGGGTTTCTGAATG	77
BCR-ABL1 R2	CGCTGAAGGGCTTTTGAACTC
BCR-ABL1 P	FAM-ATCGTCCACTCAGCCAC-MGB

均匀性	组间均方	组内均方	F值	F_0.05临界值
瓶内	7.129×10¹⁰	5.432×10¹¹	0.131	F_0.05（2,12）=3.89
瓶间	2.207×10¹¹	1.229×10¹¹	1.796	F_0.05（9,20）=2.39

均匀性	组间均方	组内均方	F值	F_0.05临界值
瓶内	7.129×10¹⁰	5.432×10¹¹	0.131	F_0.05（2,12）=3.89
瓶间	2.207×10¹¹	1.229×10¹¹	1.796	F_0.05（9,20）=2.39

温度	b₁	b₀	s（b₁）	t_0.95,n-2·s（b₁）
4 ℃	-0.063×10⁶	4.802×10⁶	0.150×10⁶	0.645×10⁶
20~25 ℃	-0.092×10⁶	6.351×10⁶	0.091×10⁶	0.391×10⁶
-20 ℃	-0.195×10⁶	5.615×10⁶	0.070×10⁶	0.301×10⁶