葡萄糖-6-磷酸脱氢酶基因突变检测国家参考品的建立

Establishment of the national reference material for the detection of glucose-6-phosphate dehydrogenase gene mutation

目的:制备葡萄糖-6-磷酸脱氢酶(G6PD)基因突变检测国家参考品,并进行协作标定。方法:收集G6PD基因变异样本30份,包含3份野生型样本和27份突变样本,并构建永生化淋巴细胞系。建系的细胞系样本进行基因组DNA提取并分装,制备成国家参考品。用5家协作单位的葡萄糖-6-磷酸脱氢酶基因突变检测试剂盒对国家参考品性能进行验证,确定准确性,并考察国家参考品在不同条件下(于37℃条件下放置4周,期间拿出反复冻融3次)的稳定性及均匀性。结果:对30份浓度为20 ng·μL^-1国家参考品的检测结果中,5家单位均能检测出国家参考品标示的结果,但有1家单位对1个样本多检出1个突变类型。经不同条件处理的国家参考品的稳定性和均匀性的结果均一致,表明满足国家参考品的要求。结论:本研究建立的国家参考品,可满足对G6PD基因突变检测的要求,可用于国内试剂盒的性能评价及临床实验室的质量评价。

Objective:To prepare and collaborative calibrate the national reference material for the detection of glucose-6-phosphate dehydrogenase(G6 PD) gene mutation.Methods:A total of 30 blood samples with G6 PD gene variants,including 3 wild-type samples and 27 mutation samples,were collected,and immortalized lymphocyte lines were constructed. The genomic DNA of the cell line samples was extracted and repacked to prepare the national reference material. The accuracy of the national reference material was verified using G6 PD gene mutation detection kits from five manufacturers. Moreover,the stability and homogeneity under different conditions(placed at 37 ℃ for 4 weeks and 3 cycles of freeze-thaw at-20 ℃ and 37 ℃) were also investigated. Results:All the 30 samples with a concentration of 20 ng·μL^-1 were detected by the five manufactuers,while 1 sample harboring one more mutation type was detected by one company. The results of stability and uniformity of national reference material processed under different conditions were consistent,indicating that they could meet the requirements of national reference material. Conclusion:The established national reference material is eligible for the detection of G6 PD gene mutation,and is applicable for the performance evaluation of domestic detection kits and quality control of clinical laboratories.