ObjectiveTo set up a novel non-invasive prenatal determination technique of fetal RhD genotype by real-time PCR examination of fetal DNA in RhD negative maternal plasma. MethodsPlasma fetal DNA was extracted by manual DNA micro-extraction method from the plasma of 22 RhD negative women (15-40 gestation week). We amplified the exons 7, 10 and intron 4 of RhD gene and sex-determining region Y gene (SRY) using real-time polymerase chain reaction. The presence of fetal DNA was confirmed by testing SRY. The results of genotyping and serum RhD status of the newborns were compared to evaluate the accuracy of the present method.ResultsCell-free fetal DNA was detected in the maternal plasma samples. Among the 19 RhD negative specimens, 14 cases had the specifically amplified exons 7, 10 and intron 4 of RhD gene and SRY gene, and the results were confirmed by serological examination of fetal umbilical blood after delivery. Among the 19 specimens, 3 cases were not detected the specifically amplified in exons 7, 10 and intron 4 of RhD gene and SRY gene, and the results were also confirmed by serological examination of fetal umbilical blood. The accuracy of the present method was 89.5%. SRY detection confirmed fetal DNA presence in maternal plasma in all boys. The other 2 cases only had specifically amplified exons 10 of RhD gene, and the results were confirmed as RhDel phenotype. Real-time PCR easily differentiated pregnant woman whose RBCs had a weak D phenotype (n=3) from truly RhD-negative patients. However, in these cases fetal RhD status cannot be determined.ConclusionReal time PCR detection of fetal DNA in RhD-negative maternal plasma is simple, quick and specific for noninvasive prenatal diagnosis of fetal RhD blood type, which can help to prevent hemolytic disease of newborns.