胎儿染色体非整倍体(T21、T18、T13)检测试剂盒(探针杂交法)的评价

Evaluation of fetal chromosome aneuploidy(T21,T18,T13) detection kit(probe hybridization)

目的应用基于单核苷酸多态性(SNP)检测方法的胎儿染色体非整倍体(21三体、18三体和13三体)国家参考品,评价胎儿染色体非整倍体(T21、T18、T13)检测试剂盒(探针杂交法)的性能。方法从国家参考品中提取血浆游离DNA。应用探针杂交捕获染色体目标片段并将其转换为DNA环,对DNA环进行滚环扩增,然后对滚环复制产物进行特异荧光标记并使用显微成像仪检测,通过比较21号、18号和13号染色体的荧光信号值计算三者之间的相对比例,从而评估T21、T18和T13的风险。结果三批试剂盒对国家参考品中的104例样本进行检测,其中10%胎儿游离DNA比例的国家阳性参考品结果均为相应染色体三体;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;5%胎儿游离DNA比例检测限参考品在9次测试中的检出率均为100%,3.5%胎儿游离DNA比例检测限参考品在9次测试中的检出率为75%^92%;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,重复性均符合国家参考品的要求。结论国家参考品具有很好的适用性,胎儿染色体非整倍体(T21、T18、T13)检测试剂盒(探针杂交法)的性能符合其要求。

Objective To evaluate the performance of fetal chromosome aneuploidy(T21,T18 and T13)detection kit(probe hybridization),the national reference materials for fetal trisomy 21,trisomy 18 and trisomy 13 of chromosomal aneuploidies detection by SNP method were used.Methods Plasma free DNA was extracted from national reference materials and hybridized to the target DNA fragments to form circular DNA complexes.The DNA circles were copied by rolling-circle-amplification(RCA),then labeled the rolling circle replication product(RCP)with specific fluorescent tags.The density of each chromosome and chromosome fragment raw ratio was acquired to assess the risks of T21,T18 and T13,which were tested and counted by microscopic imaging devices.Results 104 samples of national reference material were tested with three batches of kits,among which national positive reference material with 10%cell-free fetal DNA(cffDNA)fraction were the corresponding trisomy.National negative reference material with normal or other chromosomal abnormalities were not trisomy 21,18 and 13.The detection rate of the limited reference with 5%cffDNA fraction in 9 tests were 100%,and the detection rate of the limited reference with 3.5%cffDNA fraction in 9 tests were from 75%to 92%.The microduplication reference of chromosome 18 were all trisomy 18,while the results of other microdeletion and microduplication reference were not trisomy 21,18 and 13.The repeatability was in line with the requirement of national reference.Conclusion The national reference materials have good applicability,and the performance of fetal chromosome aneuploidy detection kit(probe hybridization)meets its requirements.