现代检验医学杂志2026，Vol.41Issue(3)：13-21,33,10.DOI:10.3969/j.issn.1671-7414.2026.03.003
急性髓系白血病CBFB::MYH11融合基因qPCR检测候选标准品的构建与性能评价
Development and Performance Evaluation of Candidate Reference Materials for qPCR Detection of the CBFB::MYH11 Fusion Gene in Acute Myeloid Leukemia
摘要
Abstract
Objective To develop candidate reference materials for the quantitative PCR(qPCR)detection of the CBFB::MYH11 fusion gene in acute myeloid leukemia(AML),thereby providing effective technical support for the standardization of clinical testing.Methods A recombinant plasmid containing the CBFB::MYH11 fusion gene and the internal reference gene ABL1(ratio 1:1)was constructed.An optimized diluent matrix was used,and candidate reference standards covering six concentration levels within the routine clinical detection range were prepared using the gravimetric method.The homogeneity and stability of the materials were evaluated in accordance with ISO 33405:2024 and ISO 17511:2020.Eight laboratories were organized to conduct a collaborative interlaboratory calibration using three brands of digital PCR instruments,and the expanded uncertainty of the calibration results was calculated(k=2).Results Using T1E0.01 buffer supplemented with 5～50 ng/µl salmon sperm DNA as the diluent,the samples remained stable at 20℃for at least 7 days(t=1.90、0.40,all P>0.05).Six concentration levels of samples were prepared using the optimized buffer(T1E0.01 containing 30 ng/µl salmon sperm DNA),with 170 replicates for each level.One-way ANOVA indicated good homogeneity among samples at each concentration level(F=0.70～1.50,all P>0.05).Stability studies showed that the sample remained stable for 4 weeks at 4℃(t=-1.45～1.82,all P>0.05)and for at least 3 months at-20℃(t=-1.83～1.35,all P>0.05),but repeated freeze-thaw cycles were not recommended(Groups A～E:t=2.43～10.78,all P<0.05).Combined calibration results indicated that the batch of reference material covered a wide linear range of 100 copies/µl to 105 copies/µl,with an average combined relative expanded uncertainty of 13%.Conclusions The homogeneity and stability evaluation results of the prepared CBFB::MYH11 fusion gene candidate reference material met the relevant standard requirements.The measurement expansion uncertainty was comparable to that of international reference materials,providing a reference basis for the preparation of reference materials for AML-related clinical molecular testing.关键词
急性髓系白血病/CBFB::MYH11融合基因/数字PCR/候选标准品Key words
acute myeloid leukemia/CBFB::MYH11 fusion gene/digital PCR/candidate reference material分类
医药卫生引用本文复制引用
王国雄,胡高峰,麻雅婷,许成山,李臣宾..急性髓系白血病CBFB::MYH11融合基因qPCR检测候选标准品的构建与性能评价[J].现代检验医学杂志,2026,41(3):13-21,33,10.基金项目
国家科技重大专项(2024ZD0523701). （2024ZD0523701）
