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

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Immunohematology

American National Red Cross

Subject: Medical Laboratory Technology

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ISSN: 0894-203X
eISSN: 1930-3955

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VOLUME 26 , ISSUE 2 (June 2010) > List of articles

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

Marcia C. Zago Novaretti / Azulamara da Silva Ruiz / Pedro Enrique Dorlhiac-Llacer / Dalton Alencar Fisher Chamone

Keywords : blood donors, genotyping, real time, PCR, blood groups, Diego blood group, population, gene frequency

Citation Information : Immunohematology. Volume 26, Issue 2, Pages 66-70, DOI: https://doi.org/10.21307/immunohematology-2019-205

License : (Transfer of Copyright)

Published Online: 12-March-2020

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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 Dia 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-Dib scarcity. Of the 4326 samples, we simultaneously tested 160 samples for Dia and Dib 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.

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