Application of real-time PCR and melting curve analysis in rapid Diego blood group genotyping

Abstract

The paucity of appropriate reagents for serologic typing of the Diego blood group antigens has prompted the development of a real-time PCR and melting curve analysis for Diego blood group genotyping. In this study, we phenotyped 4326 donor blood samples for Di a using semiautomated equipment. All 157 Di(a+) samples were then genotyped by PCR using sequence-specific primers (PCR-SSP) for DI*02 because of anti-Di b scarcity. Of the 4326 samples, we simultaneously tested 160 samples for Di a and Di b by serology, and for DI*01 and DI*02 by PCR-SSP and by real-time PCR. We used the same primers for Diego genotyping by real-time PCR and PCR-SSP. Melting curve profiles obtained using the dissociation software of the real-time PCR apparatus enabled the discrimination of Diego alleles. Of the total samples tested, 4169 blood donors, 96.4 percent (95% confidence interval [CI], 95.8-96.9%), were homozygous for DI*02 and 157, 3.6 percent (95% CI, 3.1-4.2%), were heterozygous DI*01/02. No blood donor was found to be homozygous for DI*01 in this study. The calculated DI*01 and DI*02 allele frequencies were 0.0181 (95% CI, 0.0173-0.0189) and 0.9819 (95% CI, 0.9791-0.9847), respectively, showing a good fit for the Hardy-Weinberg equilibrium. There was full concordance among Diego phenotype results and Diego genotype results by PCR-SSP and real-time PCR. DI*01 and DI*02 allele determination with SYBR Green I and thermal cycler technology are useful methods for Diego determination. The real-time PCR with SYBR Green I melting temperature protocol can be used as a rapid screening tool for DI*01 and DI*02 blood group genotyping.

Blood Group Testing

“…As already discussed, PCR-RFLP, PCR-SSCP, DNA sequencing, etc., are the common genetic tests used for blood typing. They can be used to identify rare blood types and study the inheritance and frequency of rare blood antigen genes, such as real-time fluorescent PCR (95,96). NGS is also suitable for identifying rare blood types without knowing specific antibodies (110,111).…”