Rapid Identification of Seven Common ABO Alleles by Two-Dimensional Polymerase Chain Reaction Technology

Jin Chen; Yuxia Zhan; Jun Zhang; Yang Yu; Shuang Yao; Guanghua Luo

doi:10.1159/000530013

Rapid Identification of Seven Common ABO Alleles by Two-Dimensional Polymerase Chain Reaction Technology

Transfus Med Hemother. 2023 Apr 24;50(6):502-514. doi: 10.1159/000530013. eCollection 2023 Dec.

Authors

Jin Chen^{1

2}, Yuxia Zhan^{1

3}, Jun Zhang^{1

3}, Yang Yu^{1

3}, Shuang Yao^{1

3}, Guanghua Luo^{1

3}

Affiliations

¹ Clinical Medical Research Center, The Third Affiliated Hospital of Soochow University, Changzhou, China.
² Department of Blood Transfusion, The Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University, Changzhou, China.
³ Changzhou Key Lab of Individualized Diagnosis and Treatment Associated with High Technology Research, Changzhou, China.

Abstract

Introduction: The molecular biology detection technology of the human ABO blood group system makes up for the limitations in many aspects compared with conventional serological typing technology. This study aimed to establish a new method to identify seven common ABO alleles (ABO*A1.01, ABO*A1.02, ABO*A2.01, ABO*B.01, ABO*O.01.01, ABO*O.01.02, and ABO*O.02.01) by two-dimensional polymerase chain reaction (2D PCR). 2D PCR can identify multiple target genes in a closed test tube by labeling specific primers with tags homologous to the sequence of fluorescently labeled probes, and melting curve analysis is performed after the fluorescent probes are hybridized with tag complementary sequences in PCR-specific products. In this study, 2D PCR and PCR sequence-specific primer (PCR-SSP) were combined to discriminate different alleles in a single reaction, which has the characteristics of high throughput, and compared with other typing techniques; the typing results can be obtained without additional operations.

Methods: The ABO*A1.01 allele genetic sequence was used as the reference sequence. The specific sense and antisense primers for seven common ABO alleles were designed on exons 6 and 7 according to the principle of 2D PCR and PCR-SSP. Single nucleotide polymorphism sites for identifying seven alleles were detected in FAM and HEX channels, respectively. Two hundred sixty DNA samples were enrolled for rapid ABO genotyping by 2D PCR, and 95 of them were selected for Sanger sequencing. The Kappa test was used to analyze the agreement of the methodologies.

Results: These 7 alleles each had four characteristic melting valleys at different single nucleotide polymorphism loci. A total of 15 genotypes were detected, including ABO*A1.01/A1.02, ABO*A1.01/O.01.01, ABO*A1.01/O.01.02, ABO*A1.02/A1.02, ABO*A1.02/O.01.01, ABO*A1.02/O.01.02, ABO*B.01/B.01, ABO*B.01/O.01.01, ABO*B.01/O.01.02, ABO*O.01.01/O.01.01, ABO*O.01.01/O.01.02, ABO*O.01.02/O.01.02, ABO*A1.01/B.01, ABO*A1.02/B.01, and ABO*B.01/O.01. v (containing a rare ABO*O allele, based on the sequencing results). The Kappa test showed completely consistent results for 2D PCR and Sanger sequencing (Kappa = 1).

Conclusion: The 2D PCR technique could be used for molecular typing of the ABO blood group, which was efficient, rapid, accurate, and economical.

Keywords: ABO alleles; Molecular typing; Sequencing; Single nucleotide polymorphism; Two-dimensional polymerase chain reaction.

Grants and funding

No funding was received to assist with the preparation of this manuscript. This work was supported by the Changzhou High-Level Medical Talents Training Project (No: 2016ZCLJ002).