Development of BCR-ABL1 candidate reference material and evaluation of measurement uncertainty

doi:10.3969/j.issn.1673-8640.2019.08.017

Abstract

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

CLC Number:

R446.1

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.

Figures/Tables 8

References 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