Application of droplet digital PCR combined with TaqMan real-time PCR in Dombrock blood group genotyping in Northwest China

Abstract

Background and objectives: The Dombrock blood group system is based on the DO gene. DO*A and DO*B antigens are the result of a single-nucleotide polymorphism (SNP) on this gene. The introduction of Do antigens through blood transfusion or other invasive factors like infection may result in the production of Do antibodies, which may cause serious haemolytic transfusion reactions. In this study, TaqMan real-time PCR and droplet digital PCR were used to detect rare DO*A allele, guide the search for rare DO*A allele donors, and calculate DO alleles frequencies in mixed populations in Northwest China.

Materials and methods: In this study, the highly sensitive and accurate TaqMan real-time polymerase chain reaction (PCR) method was used to detect and screen DO genotype SNPs in combination with droplet digital PCR. We also searched for rare DO*A allele donors and calculated the frequencies of DO alleles in mixed populations.

Results: A total of 1202 donor DNA samples were collected from Northwest China, of which 202 were used to detect DO allele SNPs using TaqMan real-time PCR. The rare DO*A allele was detected in the other 1000 blood donors by droplet digital PCR, and gene frequencies were inferred from dual channel droplet digital PCR data. Among 1202 donors from Northwest China, the allele frequencies of DO*A and DO*B were 0.1128 and 0.8872, respectively.

Conclusion: The sequencing results confirmed that this new way of detecting DO alleles by droplet digital PCR with specific probes can detect rare DO*A allele to predict the presence of the rare antigen Do^a and infer DO allele frequencies. This method is highly sensitive and specific.

Keywords: Dombrock blood group system; TaqMan real-time polymerase chain reaction; droplet digital polymerase chain reaction; genotype.