Abstract
Introduction: Mutations at the codon 6 of the β-globin gene (hemoglobin [Hb] S and HbC) can be routinely identified by various methods and prenatal diagnosis has been available to affected families for several years. However, the presence of maternal cells in fetal samples constitutes a serious potential source of prenatal misdiagnosis and most methods currently used to detect maternal contamination are based on the analysis of highly polymorphic loci. In addition, these methods are labor intensive and time consuming and risk carry-over contamination.
Method: We describe here a one-step method for mutation detection that uses fluorescent hybridization probes with melting curve analysis for both simultaneously prenatal diagnosis of sickle cell disease and potential maternal contamination.
Results: Retrospective and prospective prenatal diagnosis studies (conducted in 20 and 50 cases, respectively), using both the regular procedure and real-time PCR assay show perfect concordant results. We show in addition, that as little as 5% maternal contamination can be detected and that genotype determinations are unambiguous.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230: 1350–4
Embury SH, Scharf SJ, Saiki RK, et al. Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis. N Engl J Med 1987; 316: 656–61
Ghanem N, Girodon E, Vidaud M, et al. A comprehensive scanning method for rapid detection of beta-globin gene mutations and polymorphisms. Hum Mutat 1992; 1: 229–39
Waterfall CM, Cobb BD. Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis. Nucleic Acids Res 2001; 29: E119
Barany F. Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci U S A 1991; 88: 189–93
Lay MJ, Wittwer CT. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin Chem 1997; 43: 2262–7
Gundry CN, Bernard PS, Herrmann MG, et al. Rapid F508del and F508C assay using fluorescent hybridization probes. Genet Test 1999; 3: 365–70
Phillips M, Meadows CA, Huang MY, et al. Simultaneous detection of C282Y and H63D hemochromatosis mutations by dual-color probes. Mol Diagn 2000; 5: 107–16
Herrmann MG, Dobrowolski SF, Wittwer CT. Rapid beta-globin genotyping by multiplexing probe melting temperature and color. Clin Chem 2000; 46: 425–8
Moreno I, Bolufer P, Perez M, et al. Rapid detection of the major Mediterranean beta-thalassemia mutations by real-time PCR polymerase chain reaction using fluororophore-labelled hybridization probes. Br J Haematol 2002; 119: 554–7
Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997; 245: 154–60
Bernard PS, Ajioka RS, Kushner JP, et al. Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes. Am J Pathol 1998; 153: 1055–61
Bernard PS, Lay MJ, Wittwer CT. Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves. Anal Biochem 1998; 255: 101–7
Lyon E. Mutation detection using fluorescent hybridization probes and melting curve analysis. Expert Rev Mol Diagn 2001; 1: 92–101
Day NS, Tadin M, Christiano AM, et al. Rapid prenatal diagnosis of sickle cell diseases using oligonucleotide ligation assay coupled with laser-induced capillary fluorescence detection. Prenat Diagn 2002; 22: 686–91
Batanian JR, Ledbetter DH, Fenwick RG. A simple VNTR-PCR method for detecting maternal cell contamination in prenatal diagnosis. Genet Test 1998; 2: 347–50
Eichler H, Lese A, Meckies J, et al. Prenatal HLA genotyping of uncultured amniotic fluid samples contaminated with maternal blood. Am J Obstet Gynecol 2002; 186: 1366–71
Pietrapertosa A, Campanale D, Palma A, et al. Analysis of HLA-DRB1*-A* and-B* alleles in prenatal diagnosis for determination of maternal contamination in fetal DNA. Mol Hum Reprod 2002; 8: 586–8
Antoniadi T, Yapijakis C, Kaminopetros P, et al. A simple and effective approach for detecting maternal cell contamination in molecular prenatal diagnosis. Prenat Diagn 2002; 22: 425–9
Acknowledgements
No sources of funding were used to assist in conducting this study. The author has no conflicts of interest that are directly relevant to the content of this manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Costa, C., Pissard, S., Girodon, E. et al. A One-Step Real-Time PCR Assay for Rapid Prenatal Diagnosis of Sickle Cell Disease and Detection of Maternal Contamination. CNS Drugs 7, 45–48 (2003). https://doi.org/10.1007/BF03260020
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03260020