选择性磁珠纯化法结合高通量测序技术在无创产前筛查中的应用
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1.南京医科大学第一附属医院;2.南京医科大学第一附属医院 心内科

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国家自然科学基金项目(面上项目,重点项目,重大项目)


Application of fragment-selective purification method to enrich fetal cell-free DNA in noninvasive prenatal testing
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Department of Obstetrics,the first affiliated hospital with Nanjing Medical University,Nanjing

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    摘要:

    目的:探讨基于DNA片段选择性磁珠纯化法(片段选择富集法)富集胎儿游离DNA结合高通量测序技术在无创产前筛查中的应用价值。方法:选取2019年8月至2021年8月在我院行胎儿染色体非整倍体无创产前筛查的孕妇共20,868例进行回顾性分析,比较传统DNA提取纯化法(传统纯化法)和片段选择法在胎儿游离DNA提取纯化质量、无创产前筛查初次检测失败率及胎儿染色体异常筛查准确性方面的差异。结果:共有5,914名孕妇采用传统纯化法结合高通量测序检测,其中33例(0.56%)判定为胎儿浓度过低需重新抽血复检;而14,954名孕妇采用片段选择法结合高通量测序进行检测,2例(0.01%)判定为胎儿浓度过低需抽血复检,两组存在显著性差异(0.56% vs.0.01%,p<0.001)。与传统纯化法(23/5,914,0.39%)相比,片段选择法结合高通量测序检出(47/14,954,0.31%)目标染色体异常率(21三体+18三体+13三体)无明显差异(p>0.05),其中胎儿21三体(54.55% vs.64.86% p>0.05)、18三体(83.33% vs.60% p>0.05)和13三体(50.00% vs.25.00% p>0.05)阳性 预测值均无显著性差异。经介入性产前诊断及产后随访复核,传统纯化法组和片段选择法组各有1例假阴性,两组间染色体异常漏诊率无显著性差异(0.02% vs.0.007%,p>0.05)。结论:基于DNA片段选择性磁珠纯化富集法,可以富集母体外周血浆中胎源游离DNA的含量,减少因胎儿浓度过低所致重复抽血的风险,同时不影响目标染色体疾病的阳性预测值。

    Abstract:

    Objective: To explore the application of fragmental enrichment method of fetal free DNA based on carboxylated nano magnetic beads combined with high-throughput sequencing technology in non-invasive prenatal testing. Methods: A total of 20,868 pregnant women who performed non-invasive prenatal diagnosis in the prenatal diagnosis center of our hospital from August 2019 to August 2021 were enrolled. The differences between traditional purified DNA extraction method and fragmental enrichment method combined with high-throughput sequencing technology on the quality of extracted DNA and the diagnosis of fetal chromosomal abnormalities were compared. Results: A total of 5,914 pregnant women were detected by traditional purified DNA extraction method combined with high-throughput sequencing, and 33 of them (0.56%) needed to be retested due to low concentration of circulating DNA; While 14,954 pregnant women were detected by fragmental enrichment method combined with high-throughput sequencing, 2 cases (0.01%) needs repeat blood sampling for examination, and there was a significant difference in the proportion of repeated blood sampling between the two groups (0.56% vs.0.01%, p<0.001). Compared with the traditional DNA extraction method (23/5,914, 0.39%), the fragmental enrichment method combined with high-throughput sequencing (47/14,954, 0.31%) showed no significant difference in the chromosome abnormality rate (trisomy 21+trisomy 18+trisomy 13) (p>0.05). After amniocentesis and postpartum follow-up review, there was 1 case missed in each group, and there was no significant difference between the two groups (0.02% vs.0.007%, p>0.05). Particularly,compared with the traditional DNA purification method, the fragmental enrichment method showed no significant difference in the positive rate of trisomy 21 (54.55% vs.64.86% p>0.05), trisomy 18 (83.33% vs.60% p>0.05) and trisomy 13 (50.00% vs.25.00% p>0.05) in maternal peripheral blood. Conclusions: The fragmental enrichment method of fetal free DNA based on carboxylated nano magnetic beads can enrich the fetal free DNA content in maternal peripheral blood circulation, thereby reducing the risk of repeated blood drawing in pregnant women. In addition, the fragmental enrichment method combined with high-throughput sequencing will not lead to the loss of target DNA, reducing the detection rate of chromosomal abnormalities.

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  • 收稿日期:2023-04-13
  • 最后修改日期:2023-08-01
  • 录用日期:2023-10-18
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