Comparison of real-time polymerase chain reaction assay methods for detection of RHD gene in amniotic fluid

Abstract

Hemolytic disease of the newborn is the clinical condition in which Rh blood group antigens in couples are incompatible with each other and mother is negative for the antigen, whereas father is positive. Although RHD antigen encoded by RHD gene that is localized on chromosome 1 determines person's Rh genotyping, this incompatibility can lead to delivery as anemia, jaundiced, or dead in mother's uterus. In recent years, improvements have occurred in the prenatal diagnosis of Rh incompatibility. Quantitative real-time polymerase chain reaction (Real-time PCR) has been improved and determining rapidly, reliably, and sensitively has been possible. In this study, the determination of RHD genotyping was investigated using fetal DNA obtained from amniotic fluid and SYBR Green I and TaqMan probe methods were compared, and reliability in prenatal diagnosis of these methods was determined. We studied 35 pregnant women in the second trimester of pregnancy. “SYBR Green I” and “TaqMan” probes results for RHD gene of genomic DNA extracted from total 35 different amniotic fluid samples acquired from 10 RHD (-) and 25 pregnant women randomly were analyzed. DNA extracted from amniotic fluid was analyzed for RHD gene with real-time PCR and the results were then compared with the RHD fetal genotype determined on RHD phenotype of the red blood cells of the infants at birth. The results of RHD TaqMan probes PCR analysis of amniotic fluid DNA were completely concordant with the fetal blood group analysis after birth. Real-time PCR using the TaqMan probes has proven to be more sensitive, accurate, and specific for RHD gene than SYBR Green I method.