Molecular analysis of the RHD pseudogene by duplex real-time polymerase chain reaction.

OBJECTIVES: In this study, we aimed to present a strategy for the detection of the RHD pseudogene (RHDψ) based on a real-time polymerase chain reaction (PCR) assay.

BACKGROUND: The D-negative phenotype is associated with many genetic alterations. In populations with African ancestry, this phenotype commonly results from the silent variant RHDψ. The evaluation of RHDψ is essential for correct inference of the RhD phenotype in order to avoid false-positive results.

METHODS: We utilised a new method for the simultaneous detection of RHDψ and a fragment from exon 5 of the wild-type RHD gene based on duplex real-time PCR assay.

RESULTS: The PCR assay allowed specific detection of RHDψ. There was complete agreement between the results generated by the new test and the results generated by molecular analysis performed using end-point PCR methods previously described.

CONCLUSIONS: The assay developed is easy to execute and presents the potential for routine use at blood banks and other associated facilities where it is desired to determine the presence of RHDψ.