To determine the prevalence gene pseudogene and hybrid gene and standardization of molecular techniques in method of Taqman on real-time PCR for RHD genotyping.
203 samples of D-negative donor were used to establish and validate the effectiveness of RHD genotyping in real-time PCR using Taqman technology. The extraction was performed using a commercial kit QIAmp DNA mini kit. Samples exon 10 and 7 positive were submitted to amplification of exon 5, confirming the pseudogene RHD¦·, whereas exon 10 + exon 7 - for the hybrid gene (C) cdes and mutation C733G (Leu245Val) of the RHCE gene.
Twenty-five (12.3 % ) samples were positive, 14 amplified for both exons 10 and 7 while in 11 only for the exon 10. When extended the screening using exon 10, 7 and 5, only 06 amplified. The pseudogene was present in 07 samples (3.5 % ) and the hybrid RHD-CE (3-7) in 04 (1.97 % ), while in 177 (87.2 % ) of Rh negative donors were RHD gene deletion. In 07 samples not amplified for exon 3 had mutated and the mutation C733G antigen.
The prevalence of pseudogene was 3.5 % and the gene hybrid RHD-CE of 1.9 % . This approach for real-time PCR as a complementary tool is technically feasible and the results of this study helped develop a new strategy for RHD genotyping.