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OA
Development and Performance Evaluation of Candidate Reference Materials for qPCR Detection of the CBFB::MYH11 Fusion Gene in Acute Myeloid Leukemia
目的 研制急性髓系白血病(AML)CBFB::MYH11融合基因qPCR检测候选标准品,为临床检测标准化提供有效技术支撑.方法 构建包含CBFB::MYH11融合基因和内参基因ABL1(比值1:1)的重组质粒,优化稀释液基质,通过重量法制备覆盖常规临床检测范围的6个浓度水平候选标准品.依据ISO33405:2024和ISO17511:2020评估候选标准品的均匀性与稳定性,并组织8家实验室使用3种品牌数字PCR仪联合定值,计算定值结果的扩展不确定度(k=2).结果 以添加5～50ng/µl鲑鱼精DNA的T1E0.01缓冲液作为稀释液,样品在20℃下可稳定保存至少7天(t=1.90、0.40,均P>0.05).以优化后缓冲液(添加30ng/µl鲑鱼精DNA的T1E0.01缓冲液)制备了6个浓度水平样品,各170支.单因素方差分析显示各浓度水平样品的均匀性良好(F=0.70～1.50,均P>0.05).稳定性研究显示,该样品在4℃下可稳定保存4周(t=-1.45～1.82,均P>0.05),在-20℃下可稳定保存至少3个月(t=-1.83～1.35,均P>0.05),但禁忌反复冻融(A～E组:t=2.43～10.78,均P<0.05).联合定值结果显示该批标准品可覆盖100～105拷贝/µl的宽线性范围,合成相对扩展不确定度平均值为13%.结论 制备的CBFB::MYH11融合基因候选标准品的均匀性和稳定性结果符合相关标准要求,量值扩展不确定度接近国际标物水平,可为AML相关临床分子检测的标准品制备提供参考依据.
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.
王国雄;胡高峰;麻雅婷;许成山;李臣宾
北京医院/国家老年医学中心,国家卫生健康委临床检验中心,中国医学科学院老年医学研究院,北京 100730||北京协和医学院/中国医学科学院,北京 100730北京医院/国家老年医学中心,国家卫生健康委临床检验中心,中国医学科学院老年医学研究院,北京 100730北京医院/国家老年医学中心,国家卫生健康委临床检验中心,中国医学科学院老年医学研究院,北京 100730北京医院/国家老年医学中心,国家卫生健康委临床检验中心,中国医学科学院老年医学研究院,北京 100730北京医院/国家老年医学中心,国家卫生健康委临床检验中心,中国医学科学院老年医学研究院,北京 100730||北京协和医学院/中国医学科学院,北京 100730
医药卫生
急性髓系白血病CBFB::MYH11融合基因数字PCR候选标准品
acute myeloid leukemiaCBFB::MYH11 fusion genedigital PCRcandidate reference material
《现代检验医学杂志》 2026 (3)
13-21,33,10
国家科技重大专项(2024ZD0523701).
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