胎儿染色体非整倍体21三体、18三体和13三体检测试剂盒(高通量测序法)行业标准的制定

Establishment of industry standards for fetal chromosome aneuploidy including trisomy 21,trisomy 18 and trisomy 13(Next-Generation Sequencing)

目的制定胎儿染色体非整倍体21三体、18三体和13三体检测试剂盒(高通量测序法)行业标准,使用BioelectronSeq 4000等不同测序平台的试剂盒进行验证,评价标准的可行性。方法使用高通量测序用外周血胎儿染色体非整倍体(T21、T18和T13)国家参考品,按照拟定行业标准的要求进行验证。首先提取国家参考品的血浆游离DNA,制备文库,上机测序,通过生物信息软件分析获得染色体数目的结果。结果胎儿游离DNA浓度为10%和5%的国家阳性参考品结果均为相应染色体三体;40个3.5%浓度的国家检测限参考品,其中至少20个参考品结果为相应染色体三体;嵌合比例为70%和30%的国家嵌合体参考品均为相应染色体三体;国家微缺失微重复参考品中18号染色体微重复的参考品均为18三体,其余参考品结果均不是21、18和13三体;染色体正常或其他染色体异常的国家阴性参考品,结果均不是21、18和13三体。结论制定的行业标准可以用于该类产品试剂盒的质量评价,为产品注册检验及上市后监督管理提供依据。

Objective To develop,verify,and evaluate the feasibility of industry standards for diagnosing fetal chromosome aneuploidy trisomy 21,trisomy 18,and trisomy 13 using next generation sequencing(NGS).Methods According to the requirements of the proposed industry standards and national reference materials of fetal chromosomal aneuploidy abnormality(T21,T18 and T13),cell-free DNA in plasma was sequenced using NGS.Chromosome number was determined using bioinformatics analysis software.Results National positive references with the fetal cell-free DNA concentrations of 10%and 5%ware the corresponding trisomy.In 40 national limit references with 3.5%concentrations,at least 20 samples ware the corresponding trisomy.National chimera references with the chimera proportion of 70%and 30%were the corresponding trisomy.Trisomy 18 was found in two samples with chromosome 18 microduplication in national microdeletion and microduplication references,while the results of other samples were not trisomy21,trisomy 18 and trisomy 13.Using national negative references employing normal chromosomes or chromosomes with other abnormalities,the results were negative for trisomy 21,18 and 13.Conclusion The established industry standards can evaluate kit quality,and provide a basis for product registration and post-marketing supervision and management.