杜氏肌营养不良基因突变检测国家参考品的研制

Development of national reference material for Duchenne muscular dystrophy gene mutation detection

目的建立杜氏肌营养不良基因(DMD)突变检测国家参考品。方法收集DMD突变患儿及其家系血清,对建系成功的46份细胞系扩增繁殖至所需量后,进行基因组DNA提取并分装,制备成国家参考品。8家实验室采用8种二代测序(NGS)试剂和2种多重连接依赖性探针扩增试剂(MLPA)对候选参考品进行了验证。通过NGS和MLPA试剂检测候选参考品评价参考品的均匀性。通过NGS试剂对反复冻融3次后的候选参考品进行检测以评价稳定性。结果4家实验室采用MLPA试剂的检测结果一致,8家实验室采用NGS试剂的检测结果基本一致,对于突变位点信息有争议的参考品,采用一代测序进行了测序验证。NGS和MLPA各自均匀性检测结果均一致,反复冻融3次后的检测结果与常规保存的检测结果一致。结论通过协作验证以及均匀性、稳定性分析,成功建立了DMD突变检测国家参考品,该参考品可用于DMD基因突变检测试剂盒的性能评价。

Objective To establish a national reference for the detection of Duchenne muscular dystrophy(DMD)gene mutation.Methods Serum samples from children with DMD gene mutation and their families were collected,and 46 successfully established cell lines were expanded and multiplied to the required amount.Genomic DNA was extracted and aliquoted to prepare national reference materials.The candidate reference was validated with 8 next⁃generation sequencing(NGS)kits and 2 multiplex ligation dependent probe amplification(MLPA)kits from 8 manufacturers.The candidate reference was detected by the NGS kit and MLPA kit to evaluate the homogeneity.The stability of the candidate reference was evaluated by the NGS kit after repeated freezing and thawing three times.Results The results of four laboratories using MLPA kits were consistent,and the results of eight laboratories using NGS kits were basically the same.For the candidate reference with controversial mutation site information,the first⁃generation sequencing was used for sequencing verification.The homogeneity test results of NGS and MLPA were consistent,and the stability results were consistent with the results of conventional preservation.Conclusion Through collaborative verification,homogeneity and stability analysis,the national reference for DMD gene mutation detection was successfully established,which can be used for performance evaluation of DMD gene mutation detection kits.