A one-step real-time PCR assay for rapid prenatal diagnosis of sickle cell disease and detection of maternal contamination.

INTRODUCTION: Mutations at the codon 6 of the beta-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.