Prenatal diagnosis of genetic diseases directly using paper-dried cord blood as the starting material for PCR

  • Huan HuangEmail author
  • You Zhou
  • Jiajia Zhang
  • Weiyin Yao
  • Guoying ZhangEmail author
Research Paper


A rapid and low-cost method of diagnosis is becoming important for detecting fetal inherited diseases, including single-gene disorders and chromosomal abnormalities. Here, we demonstrated an innovation that use paper-dried cord blood (PCB) as the starting material for PCR and whole genome amplification without any DNA extraction step at a very low cost. A novel PCR buffer named “DDB buffer” containing ammonium sulfate and glycerol were used instead of the conventional 10× PCR buffer. The amplicons were directly analyzed through microchip electrophoresis and whole genome sequencing. Inhibitory substances in filter paper were effectively inactivated using DDB buffer. Direct PCR amplification of DNA fragments ranging from 100 to 900 bp using filter paper spotted with 0.5 to 5 μL of cord blood and various anticoagulants was successful. We were able to determine fetal single-gene disorders and chromosomal diseases in all 46 chromosomes using PCB samples successfully. Compared with prenatal diagnosis using purified DNA, the proposed method is simple, fast, less prone to cross-contamination at minimal cost. Researchers and clinical and healthcare workers may employ this method for genetic diagnosis using cord blood samples with minimum laboratory resources. This method is very promising for a variety of genetic diagnosis applications in underserved communities at the point of need in developing areas.

Graphical abstract


Direct PCR Paper-dried cord blood Prenatal diagnosis Single-gene disorders Chromosomal abnormalities 



Collagen type 1 alpha 2


Developmental delay


Duchenne muscular dystrophy


Intellectual disability




Phenylalanine hydroxylase


Paper-dried cord blood


Polymerase chain reaction


Survival of motor neuron 1


Funding information

This work was supported by the National Natural Science Foundation of China (81741040), Medical Talent of Empowering Medicine through Science and Education Program (QNRC2016618), the Key Provincial Talents of Maternal and Child Health Program in Jiangsu Province (FRC2017-43), and the project for the Priority Academic Program Development of Jiangsu Higher Education Institutions (JX10231802). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

216_2019_2048_MOESM1_ESM.pdf (153 kb)
ESM 1 (PDF 152 kb)


  1. 1.
    Van den Veyver IB, Roa BB. Applied molecular genetic techniques for prenatal diagnosis. Curr Opin Obstet Gynecol. 1998;10(2):97–103. Scholar
  2. 2.
    Hern WM. Fetal diagnostic indications for second and third trimester outpatient pregnancy termination. Prenat Diagn. 2014;34(5):438–44. Scholar
  3. 3.
    Altarescu G. Prevention is the best therapy: the geneticist’s approach. Pediatr Endocrinol Rev. 2016;13(Suppl 1):649–54. Scholar
  4. 4.
    Liu L, Zhou P, Cao Z, Tan X. Middle pregnancy ultrasound screening for fetal chromosomal diseases. Mol Med Rep. 2017;16(5):7641–8. Scholar
  5. 5.
    Duan Y, Li Y, Xue Q. Serological prenatal screening and diagnosis for Down syndrome. Clin Exp Obstet Gynecol. 2014;41(5):572–4. Scholar
  6. 6.
    Xiao H, Yang YL, Zhang CY, Liao EJ, Zhao HR, Liao SX. Karyotype analysis with amniotic fluid in 12365 pregnant women with indications for genetic amniocentesis and strategies of prenatal diagnosis. J Obstet Gynaecol. 2016;36(3):293–6. Scholar
  7. 7.
    Pietropolli A, Martelli F, Vicario R, Montagnoli C, Ticconi C, Piccione E. Evaluation of fetal heart rate variation during amniocentesis: correlation with fetal karyotype. J Matern Fetal Neonatal Med. 2011;24(4):587–9. Scholar
  8. 8.
    Liu YH, Xie RG, Zhang XY, Wei SD, He Y, Xu WF, et al. A new partial trisomy 12p with artery catheter vagus, congenital cataracts, external auditory canal, and no turbinate. Gene. 2012;509(1):164–7. Scholar
  9. 9.
    Shalev SA, Shalev E, Pras E, Shneor Y, Gazit E, Yaron Y, et al. Evidence for blood chimerism in dizygotic spontaneous twin pregnancy discordant for Down syndrome. Prenat Diagn. 2006;26(9):782–4. Scholar
  10. 10.
    Brison N, Van Den Bogaert K, Dehaspe L, van den Oever JM, Janssens K, Blaumeiser B, et al. Accuracy and clinical value of maternal incidental findings during noninvasive prenatal testing for fetal aneuploidies. Genet Med. 2017;19(3):306–13. Scholar
  11. 11.
    Hudecova I, Jiang P, Davies J, Lo YMD, Kadir RA, Chiu RWK. Noninvasive detection of F8 int22h-related inversions and sequence variants in maternal plasma of hemophilia carriers. Blood. 2017;130(3):340–7. Scholar
  12. 12.
    Hui WW, Jiang P, Tong YK, Lee WS, Cheng YK, New MI, et al. Universal haplotype-based noninvasive prenatal testing for single gene diseases. Clin Chem. 2017;63(2):513–24. Scholar
  13. 13.
    Sahoo T, Dzidic N, Strecker MN, Commander S, Travis MK, Doherty C, et al. Comprehensive genetic analysis of pregnancy loss by chromosomal microarrays: outcomes, benefits, and challenges. Genet Med. 2017;19(1):83–9. Scholar
  14. 14.
    Zou Z, Huang L, Lin S, He Z, Zhu H, Zhang Y, et al. Prenatal diagnosis of posterior fossa anomalies: additional value of chromosomal microarray analysis in fetuses with cerebellar hypoplasia. Prenat Diagn. 2017;38(2):91–8. Scholar
  15. 15.
    Srebniak MI, Knapen M, Polak M, Joosten M, Diderich KEM, Govaerts LCP, et al. The influence of SNP-based chromosomal microarray and NIPT on the diagnostic yield in 10,000 fetuses with and without fetal ultrasound anomalies. Hum Mutat. 2017;38(7):880–8. Scholar
  16. 16.
    Poulou M, Destouni A, Kakourou G, Kanavakis E, Tzetis M. Prenatal diagnosis for CF using high resolution melting analysis and simultaneous haplotype analysis through QF-PCR. J Cyst Fibros. 2014;13(6):617–22. Scholar
  17. 17.
    Sun L, Fan Z, Long J, Weng X, Tang W, Pang W. Rapid prenatal diagnosis of aneuploidy for chromosomes 21, 18, 13, X, and Y using segmental duplication quantitative fluorescent PCR (SD-QF-PCR). Gene. 2017;627:72–8. Scholar
  18. 18.
    Orhant L, Anselem O, Fradin M, Becker PH, Beugnet C, Deburgrave N, et al. Droplet digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia. Prenat Diagn. 2016;36(5):397–406. Scholar
  19. 19.
    Bili C, Divane A, Apessos A, Konstantinos T, Apostolos A, Ioannis B, et al. Prenatal diagnosis of common aneuploidies using quantitative fluorescent PCR. Prenat Diagn. 2002;22(5):360–5. Scholar
  20. 20.
    Huang H, Li S, Lu S, Ge H, Sun L. Prenatal diagnosis of single gene disorders using amniotic fluid as the starting material for PCR. Analyst. 2016;141(1):285–90. Scholar
  21. 21.
    Bu Y, Huang H, Zhou G. Direct polymerase chain reaction (PCR) from human whole blood and filter-paper-dried blood by using a PCR buffer with a higher pH. Anal Biochem. 2008;375(2):370–2. Scholar
  22. 22.
    Gemperle-Britschgi C, Iorgulescu D, Mager MA, Anton-Paduraru D, Vulturar R, Thony B. A novel common large genomic deletion and two new missense mutations identified in the Romanian phenylketonuria population. Gene. 2016;576:182–8. Scholar
  23. 23.
    Aldamiz-Echevarria L, Llarena M, Bueno MA, Dalmau J, Vitoria I, Fernandez-Marmiesse A, et al. Molecular epidemiology, genotype-phenotype correlation and BH4 responsiveness in Spanish patients with phenylketonuria. J Hum Genet. 2016;61(8):731–44. Scholar
  24. 24.
    Kosuga M, Mashima R, Hirakiyama A, Fuji N, Kumagai T, Seo JH, et al. Molecular diagnosis of 65 families with mucopolysaccharidosis type II (Hunter syndrome) characterized by 16 novel mutations in the IDS gene: genetic, pathological, and structural studies on iduronate-2-sulfatase. Mol Genet Metab. 2016;118(3):190–7. Scholar
  25. 25.
    Brusius-Facchin AC, Abrahao L, Schwartz IV, Lourenco CM, Santos ES, Zanetti A, et al. Extension of the molecular analysis to the promoter region of the iduronate 2-sulfatase gene reveals genomic alterations in mucopolysaccharidosis type II patients with normal coding sequence. Gene. 2013;526(2):150–4. Scholar
  26. 26.
    Wang Y, Cui Y, Zhou X, Han J. Development of a high-throughput resequencing array for the detection of pathogenic mutations in osteogenesis imperfecta. PLoS One. 2015;10(3):e0119553. Scholar
  27. 27.
    Quist EM, Doan R, Pool RR, Porter BF, Bannasch DL, Dindot SV. Identification of a candidate mutation in the COL1A2 gene of a Chow Chow with osteogenesis imperfecta. J Hered. 2017;109(3):308–14. Scholar
  28. 28.
    Kou HS, Wang CC. Molecular inversion probes equipped with discontinuous rolling cycle amplification for targeting nucleotide variants: determining SMN1 and SMN2 genes in diagnosis of spinal muscular atrophy. Anal Chim Acta. 2017;977:65–73. Scholar
  29. 29.
    Taylor JL, Lee FK, Yazdanpanah GK, Staropoli JF, Liu M, Carulli JP, et al. Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency. Clin Chem. 2015;61(2):412–9. Scholar
  30. 30.
    Liu S, Song L, Cram DS, Xiong L, Wang K, Wu R, et al. Traditional karyotyping vs copy number variation sequencing for detection of chromosomal abnormalities associated with spontaneous miscarriage. Ultrasound Obstet Gynecol. 2015;46(4):472–7. Scholar
  31. 31.
    Chard RL, Norton ME. Genetic counseling for patients considering screening and diagnosis for chromosomal abnormalities. Clin Lab Med. 2016;36(2):227–36. Scholar
  32. 32.
    Liang D, Lv W, Wang H, Xu L, Liu J, Li H, et al. Non-invasive prenatal testing of fetal whole chromosome aneuploidy by massively parallel sequencing. Prenat Diagn. 2013;33(5):409–15. Scholar
  33. 33.
    Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754–60. Scholar
  34. 34.
    Wang Y, Chen Y, Tian F, Zhang J, Song Z, Wu Y, et al. Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with noninvasive prenatal testing. Clin Chem. 2014;60(1):251–9. Scholar
  35. 35.
    Al-Soud WA, Jonsson LJ, Radstrom P. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol. 2000;38(1):345–50 Scholar
  36. 36.
    Al-Soud WA, Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol. 2001;39(2):485–93. Scholar
  37. 37.
    Deter RL, Lee W, Kingdom JCP, Romero R. Fetal growth pathology score: a novel ultrasound parameter for individualized assessment of third trimester growth abnormalities. J Matern Fetal Neonatal Med. 2018;31(7):866–76. Scholar
  38. 38.
    Khalil A, Bennet S, Thilaganathan B, Paladini D, Griffiths P, Carvalho JS. Prevalence of prenatal brain abnormalities in fetuses with congenital heart disease: a systematic review. Ultrasound Obstet Gynecol. 2016;48(3):296–307. Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Obstetrics and GynecologyThe First Affiliated Hospital of Nanjing Medical UniversityNanjingChina
  2. 2.Department of Department of Cell Systems & AnatomyUniversity of Texas Health Science Center at San AntonioSan AntonioUSA

Personalised recommendations