胎儿游离DNA片段富集技术在无创产前检测技术中的研究进展

doi:10.12280/gjszjk.20250564

摘要/Abstract

摘要：

胎儿游离DNA（cell-free fetal DNA，cffDNA）是无创产前检测技术（noninvasive prenatal test，NIPT）的核心生物标志物，其在母体血浆中丰度低且其母源背景干扰强，严重制约了NIPT的敏感度与适用范围。基于cffDNA相较于母体游离DNA（cell-free DNA，cfDNA）具有片段长度更短的特点，多种片段长度富集技术相继发展，从早期凝胶电泳与磁珠尺寸筛选，到聚合酶链反应（polymerase chain reaction，PCR）突变测序、计算机模拟方法及微流控制系统，均显著提升了cffDNA的分离效率与纯度。临床研究表明，富集胎儿分数（fetal fraction，FF）后，在低FF、高龄、肥胖及妊娠早期等高风险人群中，NIPT对常见三体综合征的敏感度和特异度均显著增强，同时可降低测序深度需求，提升成本效益。未来，cffDNA片段富集技术的研究将朝着进一步提高富集效率、降低成本、简化操作流程、扩大临床应用范围、增加多技术联合的方向发展。

关键词: 游离核酸, 非侵入性产前检测, 聚合酶链反应, 胎儿游离DNA, 胎儿分数

Abstract:

Cell-free fetal DNA (cffDNA) serves as the core biomarker for noninvasive prenatal test (NIPT), yet its low abundance in maternal plasma and the substantial maternal background interference significantly limit the sensitivity and applicability of NIPT. Leveraging the characteristic shorter fragment length of cffDNA compared to maternal cell-free DNA (cfDNA), various fragment length-based enrichment techniques have been developed, ranging from early methods such as gel electrophoresis and magnetic bead size selection to polymerase chain reaction (PCR)-based mutation sequencing, computational simulation approaches, and microfluidic systems. The technique development has markedly improved the separation efficiency and purity of cffDNA. Clinical studies demonstrate that after enrichment of the fetal fraction (FF), NIPT exhibits significantly enhanced sensitivity and specificity for common trisomy syndromes in high-risk populations (including those with low FF, advanced maternal age, obesity, and early pregnancy) while also reducing sequencing depth requirements and improving cost-effectiveness. Future research on cffDNA fragment enrichment technology will continue to evolve toward further increasing enrichment efficiency, lowering costs, simplifying operational procedures, expanding clinical applications, and promoting the integration of multiple techniques.

Key words: Cell-free nucleic acids, Noninvasive prenatal testing, Polymerase chain reaction, Cell-free fetal DNA, Fetal fraction

安清莎, 崔瑞芳, 黄雨霄, 陶逸伦, 李晓泽. 胎儿游离DNA片段富集技术在无创产前检测技术中的研究进展[J]. 国际生殖健康/计划生育杂志, 2026, 45(2): 139-144.

AN Qing-sha, CUI Rui-fang, HUANG Yu-xiao, TAO Yi-lun, LI Xiao-ze. Research Advances in Enrichment Technology of Cell-Free Fetal DNA Fragment for Noninvasive Prenatal Test[J]. Journal of International Reproductive Health/Family Planning, 2026, 45(2): 139-144.

参考文献 40

[1]	Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum[J]. Lancet, 1997, 350(9076):485-487. doi: 10.1016/S0140-6736(97)02174-0.
[2]	Xu C, Li J, Chen S, et al. Genetic deconvolution of fetal and maternal cell-free DNA in maternal plasma enables next-generation non-invasive prenatal screening[J]. Cell Discov, 2022, 8(1):109. doi: 10.1038/s41421-022-00457-4. pmid: 36229437
[3]	Welker NC, Lee AK, Kjolby R, et al. High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening[J]. Genet Med, 2021, 23(3):443-450. doi: 10.1038/s41436-020-01009-5. pmid: 33190143
[4]	Deng C, Liu S. Factors Affecting the Fetal Fraction in Noninvasive Prenatal Screening: A Review[J]. Front Pediatr, 2022, 10:812781. doi: 10.3389/fped.2022.812781.
[5]	Hanson B, Scotchman E, Chitty LS, et al. Non-invasive prenatal diagnosis (NIPD): how analysis of cell-free DNA in maternal plasma has changed prenatal diagnosis for monogenic disorders[J]. Clin Sci(Lond), 2022, 136(22):1615-1629. doi: 10.1042/CS20210380.
[6]	Lo Y, Han D, Jiang P, et al. Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies[J]. Science, 2021, 372(6538):eaaw3616. doi: 10.1126/science.aaw3616.
[7]	Zhang M, Li K, Qu S, et al. Integrative analyses of maternal plasma cell-free DNA nucleosome footprint differences reveal chromosomal aneuploidy fetuses gene expression profile[J]. J Transl Med, 2022, 20(1):536. doi: 10.1186/s12967-022-03735-7. pmid: 36401256
[8]	Ding SC, Lo Y. Cell-Free DNA Fragmentomics in Liquid Biopsy[J]. Diagnostics(Basel), 2022, 12(4):978. doi: 10.3390/diagnostics12040978.
[9]	Zhou Q, Kang G, Jiang P, et al. Epigenetic analysis of cell-free DNA by fragmentomic profiling[J]. Proc Natl Acad Sci U S A, 2022, 119(44):e2209852119. doi: 10.1073/pnas.2209852119.
[10]	Akbariqomi M, Heidari R, Gargari SS, et al. Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma[J]. J Assist Reprod Genet, 2019, 36(5):1029-1038. doi: 10.1007/s10815-019-01425-w.
[11]	Toft C, Ingerslev HJ, Kesmodel US, et al. Cell-based non-invasive prenatal testing for monogenic disorders: confirmation of unaffected fetuses following preimplantation genetic testing[J]. J Assist Reprod Genet, 2021, 38(8):1959-1970. doi: 10.1007/s10815-021-02104-5.
[12]	van de Looij A, Singh R, Hatt L, et al. Do fetal extravillous trophoblasts circulate in maternal blood postpartum?[J]. Acta Obstet Gynecol Scand, 2020, 99(6):751-756. doi: 10.1111/aogs.13880.
[13]	Chen Y, Liu Y, Shi Y, et al. Magnetic particles for integrated nucleic acid purification, amplification and detection without pipetting[J]. Trends Analyt Chem, 2020, 127:115912. doi: 10.1016/j.trac.2020.115912.
[14]	He G, Wang W, Zhou Y, et al. Ampholytic ion-exchange magnetic beads: a promising tool for selecting short fragments in circulating cell-free DNA analysis[J]. Front Oncol, 2024, 14:1397680. doi: 10.3389/fonc.2024.1397680.
[15]	Zhang B, Zhao S, Wan H, et al. High-resolution DNA size enrichment using a magnetic nano-platform and application in non-invasive prenatal testing[J]. Analyst, 2020, 145(17):5733-5739. doi: 10.1039/d0an00813c. pmid: 32748914
[16]	Liu Y, Cheng L, Wang G, et al. A nano-magnetic size selective cfDNA extraction platform for liquid biopsy with enhanced precision[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2022, 1199:123236. doi: 10.1016/j.jchromb.2022.123236.
[17]	曾雯, 祝建疆, 戚红, 等. 超顺磁性纯化磁珠筛选胎儿游离DNA技术在无创产前筛查中的应用比较[J]. 中华医学遗传学杂志, 2024, 41(7):797-802. doi: 10.3760/cma.j.cn511374-20230508-00268.
[18]	Hu P, Liang D, Chen Y, et al. An enrichment method to increase cell-free fetal DNA fraction and significantly reduce false negatives and test failures for non-invasive prenatal screening: a feasibility study[J]. J Transl Med, 2019, 17(1):124. doi: 10.1186/s12967-019-1871-x. pmid: 30975179
[19]	Hapsianto BN, Kojima N, Kurita R, et al. Direct Capture and Amplification of Small Fragmented DNAs Using Nitrogen-Mustard-Coated Microbeads[J]. Anal Chem, 2022, 94(21):7594-7600. doi: 10.1021/acs.analchem.2c00531. pmid: 35578745
[20]	Liang B, Li H, He Q, et al. Enrichment of the fetal fraction in non-invasive prenatal screening reduces maternal background interference[J]. Sci Rep, 2018, 8(1):17675. doi: 10.1038/s41598-018-35738-0. pmid: 30518878
[21]	Xue Y, Zhao G, Qiao L, et al. Sequencing Shorter cfDNA Fragments Decreases the False Negative Rate of Non-invasive Prenatal Testing[J]. Front Genet, 2020, 11:280. doi: 10.3389/fgene.2020.00280. pmid: 32273885
[22]	Zhou J, Ouyang G, Wu L, et al. Simulated confined placental mosaicism proportion (SCPMP) based on cell-free fetal DNA fraction enrichment can reduce false-positive results in non-invasive prenatal testing[J]. Prenat Diagn, 2022, 42(8):1008-1014. doi: 10.1002/pd.6150.
[23]	Qiao L, Zhang B, Wu X, et al. A fetal fraction enrichment method reduces false negatives and increases test success rate of fetal chromosome aneuploidy detection in early pregnancy loss[J]. J Transl Med, 2022, 20(1):345. doi: 10.1186/s12967-022-03555-9. pmid: 35918754
[24]	农琛. 基于PNA的核酸选择性扩增技术富集孕妇血浆cffDNA的基础研究[D]. 百色: 右江民族医学院, 2023.
[25]	吴小娟, 贺权泽, 张春花, 等. 短片段胎儿DNA富集技术可提高无创产前筛查的准确性[J]. 中国优生与遗传杂志, 2024, 32(4):828-832.
[26]	Mauger F, How-Kit A, Tost J. COLD-PCR Technologies in the Area of Personalized Medicine: Methodology and Applications[J]. Mol Diagn Ther, 2017, 21(3):269-283. doi: 10.1007/s40291-016-0254-8. pmid: 28101802
[27]	Mahdi Mortazavipour M, Mahdian R, Shahbazi S. The current applications of cell-free fetal DNA in prenatal diagnosis of single-gene diseases: A review[J]. Int J Reprod Biomed, 2022, 20(8):613-626. doi: 10.18502/ijrm.v20i8.11751. pmid: 36313257
[28]	Song C, Liu Y, Fontana R, et al. Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment[J]. Nucleic Acids Res, 2016, 44(19):e146. doi: 10.1093/nar/gkw650.
[29]	Lun FM, Tsui NB, Chan KC, et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma[J]. Proc Natl Acad Sci U S A, 2008, 105(50):19920-19925. doi: 10.1073/pnas.0810373105.
[30]	Byrou S, Makrigiorgos GM, Christofides A, et al. Fast Temperature-Gradient COLD PCR for the enrichment of the paternally inherited SNPs in cell free fetal DNA; an application to non-invasive prenatal diagnosis of β-thalassaemia[J]. PLoS One, 2018, 13(7):e0200348. doi: 10.1371/journal.pone.0200348.
[31]	Suwannakhon N, Pangeson T, Seeratanachot T, et al. Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique[J]. Hematol Rep, 2019, 11(3):8124. doi: 10.4081/hr.2019.8124.
[32]	Tan Y, Jian H, Zhang R, et al. Applying amplification refractory mutation system technique to detecting cell-free fetal DNA for single-gene disorders purpose[J]. Front Genet, 2023, 14:1071406. doi: 10.3389/fgene.2023.1071406.
[33]	Skanderup A, Zhu G, Rahman CR. 1175P Universal circulating tumor DNA quantification using deep learning[J]. Ann Oncol, 2024, 35(Suppl 2):S763. doi:10.1016/j.annonc.2024.08.1235.
[34]	Minarik G, Repiska G, Hyblova M, et al. Utilization of Benchtop Next Generation Sequencing Platforms Ion Torrent PGM and MiSeq in Noninvasive Prenatal Testing for Chromosome 21 Trisomy and Testing of Impact of In Silico and Physical Size Selection on Its Analytical Performance[J]. PLoS One, 2015, 10(12):e0144811. doi: 10.1371/journal.pone.0144811.
[35]	Kucharik M, Gnip A, Hyblova M, et al. Non-invasive prenatal testing (NIPT) by low coverage genomic sequencing: Detection limits of screened chromosomal microdeletions[J]. PLoS One, 2020, 15(8):e0238245. doi: 10.1371/journal.pone.0238245.
[36]	Guo W, Hu Y, Wang W, et al. Assessment of plasma cell-free DNA fragmentation for multi-cancer early detection: An independent clinical validation study[J]. J Clin Oncol, 2024, 42(16):e15037. doi: 10.1200/jco.2024.42.16_suppl.e15037.
[37]	Wei J, Zhao Z, Gao J, et al. Polyacrylamide/Phytic Acid/Polydopamine Hydrogel as an Efficient Substrate for Electrochemical Enrichment of Circulating Cell-Free DNA from Blood Plasma[J]. ACS Omega, 2020, 5(10):5365-5371. doi: 10.1021/acsomega.9b04397. pmid: 32201826
[38]	Alleva N, Eigen K, Ng D, et al. A Versatile and Efficient Method to Isolate DNA-Polymer Conjugates[J]. ACS Macro Lett, 2023, 12(9):1257-1263. doi: 10.1021/acsmacrolett.3c00371. pmid: 37656875
[39]	黄琴, 黄乐阳, 靳翔宇, 等. 注射式微流控芯片全集成核酸分析系统与精准医疗应用[J]. 中国激光, 2024, 51(9):190-199. doi: 10.3788/CJL231461.
[40]	Vong J, Jiang P, Cheng SH, et al. Enrichment of fetal and maternal long cell-free DNA fragments from maternal plasma following DNA repair[J]. Prenat Diagn, 2019, 39(2):88-99. doi: 10.1002/pd.5406.