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.

In 1990, Yamamoto et al. [1] successfully cloned the full-length complementary DNA sequence of the gene encoding glycosyltransferase ABO*A1.01, which made the use of molecular biology possible for ABO blood group typing. The molecular biology techniques to detect single nucleotide polymorphism (SNP) are suitable for ABO blood group genotyping because the SNP is the most important polymorphism pattern of ABO genetic structure [2‒4]. At present, the ABO genotyping methods based on polymerase chain reaction (PCR) have been developed, including PCR sequence-specific primer (PCR-SSP) assay [5, 6], PCR restriction fragment length polymorphism [7], PCR single-strand conformation polymorphism [8], PCR sequencing-based typing [9], and real-time PCR [10‒12]. Various methodologies have both advantages and disadvantages. Notably, most of these methods need to conduct multistep operations after PCR, such as enzyme digestion, transfer, and electrophoresis. The operation is cumbersome, with the risk of carrying product pollution.

The two-dimensional PCR (2D PCR) [13] can simultaneously detect multiple genes in a closed tube, not only achieving high throughput but also overcoming the time-consuming and laborious multitube detection disadvantages, as well as laboratory contamination that may be caused by open tube identification. This method is based on the base-quenched probe [14‒16] and fluorescence melting curve analysis technique [12]. A series of tags homologous to a probe sequence is printed at the 5' end of each specific primer in 2D PCR. These tag sequences are artificially designed to mutate some bases according to the probe sequence. The tag can be amplified by another primer with the amplification of a specific primer, and then the reverse-complementary sequence of the tag is recognized by the probe. The target genes can finally be identified due to the different melting temperatures (Tms) when the reverse-complementary sequences of different tags bind to one probe. Hence, high-throughput detection can be achieved by increasing the number of target genes in a channel and fluorescence channels (Fig. 1) [13]. The Tm depends on the number of tag base mutations. More tag mutations may have a melting curve at low temperature, and vice versa, while the tag that matches the probe perfectly comes out the melting curve at the highest temperature. This study aimed to combine the Tms with the PCR-SSP assay that detects SNP to identify the ABO alleles.

Fig. 1.

Schematic diagram of the basic principle of 2D PCR technology. a Base-quenched probe in which the fluorophore is labeled at the 5' end and the 3' end is phosphate-modified. b Tag sequence homologous to the probe and the dark green and light green marks on the tag are mutated bases. c Tgged primer sequence, and the tag is printed at the 5' end of primer I (orange area). d, e Amplification of the specific primer I. The blue region indicates the DNA sequence. f, g Homologous tag can be amplified with the amplification process of primer II (brown area). h Reverse-complementary sequence of the homologous tag recognized by the base quenching probe through the base complementary pairing principle during the execution of the melting curve procedure. Then, the identification of various target genes by 2D PCR is presented. Tm is taken as an X axis, and the fluorescence detection channel is taken as a Y axis. The 2D PCR technology is established in the two dimensions. By increasing the number of target genes in a channel and fluorescent channels, the purpose of high-throughput detection of target genes in a single tube can be achieved.

Fig. 1.

Schematic diagram of the basic principle of 2D PCR technology. a Base-quenched probe in which the fluorophore is labeled at the 5' end and the 3' end is phosphate-modified. b Tag sequence homologous to the probe and the dark green and light green marks on the tag are mutated bases. c Tgged primer sequence, and the tag is printed at the 5' end of primer I (orange area). d, e Amplification of the specific primer I. The blue region indicates the DNA sequence. f, g Homologous tag can be amplified with the amplification process of primer II (brown area). h Reverse-complementary sequence of the homologous tag recognized by the base quenching probe through the base complementary pairing principle during the execution of the melting curve procedure. Then, the identification of various target genes by 2D PCR is presented. Tm is taken as an X axis, and the fluorescence detection channel is taken as a Y axis. The 2D PCR technology is established in the two dimensions. By increasing the number of target genes in a channel and fluorescent channels, the purpose of high-throughput detection of target genes in a single tube can be achieved.

Close modal

Target Genes

The human ABO gene is on chromosome 9q34.1–q34.2 [17‒19] and has seven exons that span >19 kb of genomic DNA [20]. The largest part of the open reading frame is located on exons 6 and 7 [21]. The nucleotide sequences of ABO blood groups and subtypes are based on the ABO*A1.01 allele with one or more nucleotide mutations, resulting in changes in glycosyltransferase. Then, 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) have been discovered. The SNPs used to discriminate the different ABO alleles are reported in Figure 2.

Fig. 2.

Nucleotide sequence variation of seven common ABO alleles. The yellow-green background indicates the SNP loci identifying the seven alleles. Among them, c.467C>T was used as the identification point for ABO*A1.01 and ABO*A1.02 and ABO*A2.01 alleles; c.1061delC for ABO*A1.01 and ABO*A2.01 alleles; c.930G>A for ABO*A1.01 and ABO*B.01 alleles; c.261delG for ABO*A1.01 and ABO*O.01.01 and ABO*O.01.02 alleles; and c.802G>A for ABO*A1.01 and ABO*O.02.01 alleles. Meanwhile, c.261G combined with c.261delG was used to distinguish ABO*A1.01/O.01.01 and ABO*O.01.01/O.01.01 genotypes, c.297A combined with c.930G>A for ABO*A1.01/B.01 and ABO*B.01/B.01 genotypes, c.297A combined with c.646T>A for ABO*O.01.01/O.01.02 and ABO*O.01.02/O.01.02 genotypes, c.467C combined with c.467C>T for ABO*A1.01/A1.02 and ABO*A1.02/A1.02 genotypes, and c.1061C combined with c.467C>T and c.1061delC for ABO*A1.02/A2.01 and ABO*A2.01/A2.01 genotypes.

Fig. 2.

Nucleotide sequence variation of seven common ABO alleles. The yellow-green background indicates the SNP loci identifying the seven alleles. Among them, c.467C>T was used as the identification point for ABO*A1.01 and ABO*A1.02 and ABO*A2.01 alleles; c.1061delC for ABO*A1.01 and ABO*A2.01 alleles; c.930G>A for ABO*A1.01 and ABO*B.01 alleles; c.261delG for ABO*A1.01 and ABO*O.01.01 and ABO*O.01.02 alleles; and c.802G>A for ABO*A1.01 and ABO*O.02.01 alleles. Meanwhile, c.261G combined with c.261delG was used to distinguish ABO*A1.01/O.01.01 and ABO*O.01.01/O.01.01 genotypes, c.297A combined with c.930G>A for ABO*A1.01/B.01 and ABO*B.01/B.01 genotypes, c.297A combined with c.646T>A for ABO*O.01.01/O.01.02 and ABO*O.01.02/O.01.02 genotypes, c.467C combined with c.467C>T for ABO*A1.01/A1.02 and ABO*A1.02/A1.02 genotypes, and c.1061C combined with c.467C>T and c.1061delC for ABO*A1.02/A2.01 and ABO*A2.01/A2.01 genotypes.

Close modal

Plasmids

This study designed and synthesized two plasmids for methodology establishment, magnesium (Mg2+) concentration optimization, and sensitivity analysis. Plasmid 1 contained c.261delG and c.297A. Plasmid 2 contained c.467C>T, c.646T>A, c.802G>A, c.930G>A, and c.1061delC. Table 1 shows the DNA sequences of the two plasmids.

Table 1.

The DNA sequences of the plasmids

PlasmidDNA sequence*
TGC?AGT?AGG?AAG?GAT?GTC?CTC?GTG?GTAaCCC?CTT?GGC?TGG?CTC?CCA?TTG?TCT?GGG?AGG?GCA?CATTC?AAC?ATC?GAC?ATC?CTC?AAC?GAG?CAG?TTC?AGG?CTC?CAG?AAC?ACC?ACC?ATT?GGG?TTA?ACT?GTG?TTT?GC 
CACTACTATGTCTTCACCGACCAGCTGGCCGCGGTGCCCCGCGTGACGCTGGGGACCGGTCGGCAGCTGTCAGTGCTGGAGGTGCGCGCCTACAAGCGCTGGCAGGACGTGTCCATGCGCCGCATGGAGATGATCAGTGACTTCTGCGAGCGGCGCTTCCTCAGCGAGGTGGATTACCTGGTGTGCGTGGACGTGGACATGGAGATCCGCGACCACGTGGGCGTGGAGATCCTGACTCCGCTGTTCGGCACCCTGCACCCCGGCTTCTACGGAAGCAGCCGGGAGGCCTTCACCTACGAGCGCCGGCCCCAGTCCCAGGCCTACATCCCCAAGGACGAGGGCGATTTCTACTACCTGGGGAGGTTCTTCGGGGGGTCGGTGCAAGAGGTGCAGCGGCTCACCAGGGCCTGCCACCAGGCCATGATGGTCGACCAGGCCAACGGCATCGAGGCCGTGTGGCACGACGAGAGCCACCTGAACAAGTACCTACTGCGCCACAAACCCACCAAGGTGCTCTCCCCCGAGTACTTGTGGGACCAGCAGCTGCTGGGCTGGCCCGCCGTCCTGAGGAAGCTGAGGTTCACTGCGGTGCCCAAGAACCACCAGGCGGTCCGGAACCGbTGAGCGGCTGCCAGGGGCTCTGGGAGGGCTGCCGGCAGCCCCGTCCCCCTCCCGCCCTTGGTTTTAGCAGAACGG 
PlasmidDNA sequence*
TGC?AGT?AGG?AAG?GAT?GTC?CTC?GTG?GTAaCCC?CTT?GGC?TGG?CTC?CCA?TTG?TCT?GGG?AGG?GCA?CATTC?AAC?ATC?GAC?ATC?CTC?AAC?GAG?CAG?TTC?AGG?CTC?CAG?AAC?ACC?ACC?ATT?GGG?TTA?ACT?GTG?TTT?GC 
CACTACTATGTCTTCACCGACCAGCTGGCCGCGGTGCCCCGCGTGACGCTGGGGACCGGTCGGCAGCTGTCAGTGCTGGAGGTGCGCGCCTACAAGCGCTGGCAGGACGTGTCCATGCGCCGCATGGAGATGATCAGTGACTTCTGCGAGCGGCGCTTCCTCAGCGAGGTGGATTACCTGGTGTGCGTGGACGTGGACATGGAGATCCGCGACCACGTGGGCGTGGAGATCCTGACTCCGCTGTTCGGCACCCTGCACCCCGGCTTCTACGGAAGCAGCCGGGAGGCCTTCACCTACGAGCGCCGGCCCCAGTCCCAGGCCTACATCCCCAAGGACGAGGGCGATTTCTACTACCTGGGGAGGTTCTTCGGGGGGTCGGTGCAAGAGGTGCAGCGGCTCACCAGGGCCTGCCACCAGGCCATGATGGTCGACCAGGCCAACGGCATCGAGGCCGTGTGGCACGACGAGAGCCACCTGAACAAGTACCTACTGCGCCACAAACCCACCAAGGTGCTCTCCCCCGAGTACTTGTGGGACCAGCAGCTGCTGGGCTGGCCCGCCGTCCTGAGGAAGCTGAGGTTCACTGCGGTGCCCAAGAACCACCAGGCGGTCCGGAACCGbTGAGCGGCTGCCAGGGGCTCTGGGAGGGCTGCCGGCAGCCCCGTCCCCCTCCCGCCCTTGGTTTTAGCAGAACGG 

*Bold characters indicate the location of the SNP loci for the seven alleles.

aIndicating the deletion of a G before the base.

bIndicating the deletion of a C before the base.

Samples

The aim of this study was to establish a diagnostical approach to identify 7 common ABO alleles based on 2D PCR. The samples used were the residual materials of EDTA blood samples from 260 patients (118 males and 142 females; 259 cases of Chinese Han and 1 case of Thailand nationality; age range: 6–95 years) treated in the Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University from September to December 2021. The serological phenotypes of A, B, O, and AB blood groups in 260 patients were 65 cases each, and the positive and reverse typing was consistent. There were no additional interventions for patients in this study.

Apparatus and Reagents

The main experimental instruments included a real-time PCR system (SLAN-96S, China) and a fully automatic blood type analyzer (ORTHO VISION® MAX, USA). Primers, probes, and plasmids were synthesized by Shanghai Sangon Biotech Co., Ltd.

A DNA extraction kit (TIANamp Blood Kit, China) and a hot-start PCR kit were employed. The latter covers 10× Immobuffer without Mg2+ (Bioline, UK), 50 mM of magnesium chloride (MgCl2) (Bioline, UK), 2.5 mM of deoxynucleoside triphosphates (dNTPs) (Takara, China), and 5 U/µL of the IMMOLASE DNA polymerase (Bioline, UK).

Plasmid Detection Test

This study employed mixed plasmids to establish the methodology. The original concentration of plasmids 1 and 2 was ~1010 copies/µL. The two plasmids were mixed to a final concentration of ~109 copies/µL. Samples with a concentration gradient were then prepared by 1 × TE buffer according to a 10× dilution method. Finally, ~104 copies/µL mixed plasmid were selected.

Mg2+ Concentration Optimization Test

Four reaction systems with different Mg2+ concentrations of 1.0, 1.5, 2.0, and 2.5 mM were set up. The original concentration was 50 mM, and the corresponding Mg2+ amounts of the four reaction systems were 0.5, 0.75, 1.0, and 1.25 µL, respectively, based on the total amount of 25 µL in each reaction system. Here, ~104 copies/µL mixed plasmid were selected for the test.

Sensitivity Test

This study selected five diluted samples with concentrations from ~106 to ~102 copies/µL of mixed plasmids for the sensitivity test.

Serological Test

Using the ORTHO VISION® MAX Analyzer, 260 samples were tested for ABO serological phenotypes.

2D PCR Test

The ABO*A1.01 allele genetic sequence (http://www.ncbi.nlm.nih.gov/nuccore/NG_006669) was used as the reference sequence. The specific sense and antisense primers for seven common ABO alleles were designed using Primer Premier 5 on exons 6 and 7 according to the principle of 2D PCR and PCR-SSP. The tag sequences of 2D PCR and the characteristic SNP sites of each ABO allele were designed on the sense primers. SNP sites for identifying seven alleles were designed in FAM and HEX channels. The former contained c.1061delC, c.261G, c.297A, c.467C, and c.646T>A, while the latter contained c.1061C, c.261delG, c.467C>T, c.802G>A, and c.930G>A. Table 2 lists primer and probe sequences.

Table 2.

Sequences of the primers and probes

PrimerPrimer sequence (5' ? 3')c, dTm (°C)Probe sequence (5' ? 3')
c.1061delC-F* CAA?CTA?AAC?CTG?AGC?CTA?AGA?CCT?AAT?GCC?ACT?CTC?TTCCAG?GCG?GTC?CGG?AAGCG 41 FAM-CAACTAAACCTACCAACTCACATACTCTCCACTCTCTTC-P 
c.1061delC-R* CCG?TTC?TGC?TAA?AAC?CAA?GGG  
c.261G-Fa CAA?CTA?AAC?CTA?TGG?CAC?GCT?CAT?TAG?TCC?ACT?CTC?TTCAGT?AGG?AAG?GAT?GTC?CTC?GTCGTG 45 
c.297A-Fa CAA?CTA?AAC?CTA?CAC?GAG?GTA?TCG?GCC?TCC?ACT?CTC?TTCCCA?TTG?TCT?GGG?AGG?GAACA 51 
c.467C-Fb CAA?CTA?AAC?CTA?CCA?TAA?ACT?GGC?CTC?TCC?ACT?CTC?TTCACT?ACT?ATG?TCT?TCA?CCG?AACAG?CC 56 
c.646T>A-F CAA?CTA?AAC?CTA?CCA?ACT?GTT?CTA?CTC?TCC?ACT?CTC?TTCCGT?GGA?CGT?GGA?CAT?GCAGA 63 
c.646T>A-R CCG?AAC?AGC?GGA?GTC?AGG?A  
c.1061C-F* CCT?AAT?CAT?CAT?AGC?GGC?GAT?CGG?TTG?TCA?CCT?ATC?CATAGG?CGG?TCC?GCAAC?CC 37 HEX-CCTAATCATCAACCACTTACCATCACTTCACCTATCCAT-P 
c.261delG-Fa CCT?AAT?CAT?CAA?TAG?TGA?GGA?CCT?TAC?TCA?CCT?ATC?CATCAG?TAG?GAA?GGA?TGT?CCT?CGTTGTA 45 
c.261delG-Ra CAG?TTA?ACC?CAA?TGG?TGG?TGT?TCT  
c.467C>T-Fb CCT?AAT?CAT?CAA?CCA?CAA?TTT?TAA?GAT?TCA?CCT?ATC?CATCAC?TAC?TAT?GTC?TTC?ACC?GACAAGC?T 55 
c.467C>T-Rb CGC?ACC?TCC?AGC?ACT?GAC?A  
c.802G>A-F CCT?AAT?CAT?CAA?CCA?CGA?CAG?ATC?ACT?TCA?CCT?ATC?CATGGG?CGA?TTT?CTA?CTA?CCTTGGG?A 62 
c.802G>A-R GCCTCGATGCCGTTGGC  
c.930G>A-F CCT?AAT?CAT?CAA?CCA?CAC?GCC?ATC?ACT?TCA?CCT?ATC?CATCGA?GAG?CCA?CCT?GAA?CAA?GTAACTA 66 
c.930G>A-R GGG?CAC?CGC?AGT?GAA?CCT?C  
PrimerPrimer sequence (5' ? 3')c, dTm (°C)Probe sequence (5' ? 3')
c.1061delC-F* CAA?CTA?AAC?CTG?AGC?CTA?AGA?CCT?AAT?GCC?ACT?CTC?TTCCAG?GCG?GTC?CGG?AAGCG 41 FAM-CAACTAAACCTACCAACTCACATACTCTCCACTCTCTTC-P 
c.1061delC-R* CCG?TTC?TGC?TAA?AAC?CAA?GGG  
c.261G-Fa CAA?CTA?AAC?CTA?TGG?CAC?GCT?CAT?TAG?TCC?ACT?CTC?TTCAGT?AGG?AAG?GAT?GTC?CTC?GTCGTG 45 
c.297A-Fa CAA?CTA?AAC?CTA?CAC?GAG?GTA?TCG?GCC?TCC?ACT?CTC?TTCCCA?TTG?TCT?GGG?AGG?GAACA 51 
c.467C-Fb CAA?CTA?AAC?CTA?CCA?TAA?ACT?GGC?CTC?TCC?ACT?CTC?TTCACT?ACT?ATG?TCT?TCA?CCG?AACAG?CC 56 
c.646T>A-F CAA?CTA?AAC?CTA?CCA?ACT?GTT?CTA?CTC?TCC?ACT?CTC?TTCCGT?GGA?CGT?GGA?CAT?GCAGA 63 
c.646T>A-R CCG?AAC?AGC?GGA?GTC?AGG?A  
c.1061C-F* CCT?AAT?CAT?CAT?AGC?GGC?GAT?CGG?TTG?TCA?CCT?ATC?CATAGG?CGG?TCC?GCAAC?CC 37 HEX-CCTAATCATCAACCACTTACCATCACTTCACCTATCCAT-P 
c.261delG-Fa CCT?AAT?CAT?CAA?TAG?TGA?GGA?CCT?TAC?TCA?CCT?ATC?CATCAG?TAG?GAA?GGA?TGT?CCT?CGTTGTA 45 
c.261delG-Ra CAG?TTA?ACC?CAA?TGG?TGG?TGT?TCT  
c.467C>T-Fb CCT?AAT?CAT?CAA?CCA?CAA?TTT?TAA?GAT?TCA?CCT?ATC?CATCAC?TAC?TAT?GTC?TTC?ACC?GACAAGC?T 55 
c.467C>T-Rb CGC?ACC?TCC?AGC?ACT?GAC?A  
c.802G>A-F CCT?AAT?CAT?CAA?CCA?CGA?CAG?ATC?ACT?TCA?CCT?ATC?CATGGG?CGA?TTT?CTA?CTA?CCTTGGG?A 62 
c.802G>A-R GCCTCGATGCCGTTGGC  
c.930G>A-F CCT?AAT?CAT?CAA?CCA?CAC?GCC?ATC?ACT?TCA?CCT?ATC?CATCGA?GAG?CCA?CCT?GAA?CAA?GTAACTA 66 
c.930G>A-R GGG?CAC?CGC?AGT?GAA?CCT?C  

*c.1061delC-R shared by c.1061C-F and c.1061delC-F.

ac.261delG-R shared by c.261G-F, c.297A-F, and c.261delG-F.

bc.467C>T-R shared by c.467C-F and c.467C>T-F.

cThe underlined base in the primer sequence is an artificial mutation point designed according to the principle of PCR-SSP.

dThe tag sequences are highlighted in bold font, which are homologous to the probe sequences.

The optimized 2D multiplex PCR system included 2.5 µL of 10× Immobuffer, 0.5 µL of 50 mM of MgCl2, 1 µL of 2.5 mM of dNTPs, 0.5 µL of 5 U/µL of IMMOLASE DNA polymerase, 0.4 µL of each 10 µM of the probe, 0.1 µL of each 10 µM of the primer with tag (0.15 µL for c.467C and c.646T>A, 0.2 µL for c.467C>T and c.930G>A), 0.6 µL of the 10 µM primer without tag, deionized water to reach a total volume of 23 µL, and, finally, 2 µL of the mixed plasmid or the DNA template (50–200 ng/µL). The PCR programs for the hot-start reaction system were as follows: predenaturation at 95°C for 10 min to activate the DNA polymerase. The amplification process consisted of 5 cycles of 95°C for 20 s and 59°C for 15 s and 38 cycles of 95°C for 20 s, 72°C for 1 s, and 60°C for 15 s. The melting curve analysis program for the amplified product was 95°C for 1 min, from 30°C for 4 min, and then gradually increases to 70°C (temperature conversion rate: 0.06°C/s, continuously collecting fluorescence data). The final step was cooling at 40°C for 10 s.

Sanger Sequencing Analysis

The seven ABO alleles by 2D PCR were compared with the Sanger sequencing to verify its identification accuracy. Of the 260 samples, 95 were selected for the Sanger sequencing, which covered all the detected blood group genotypes by our established method. Type A had 33 cases, B had 23, O had 24, and AB had 15. Two pairs of primers were designed due to the limitation of the length of one-time sequencing (Table 3).

Table 3.

Sequences of the sequencing primers

PrimerPrimer sequence (5' ? 3')Sequence length (bp)
261–297-F* GCG?TCT?CTT?GTT?TCC?TGT?CCC 512 
261–297-R* GAC?TTA?CTT?CTT?GAT?GGC?AAA?CAC?AG 
467–1061-Fa TAC?GTG?GCT?TTC?CTG?AAG?CTG?T 789 
467–1061-Ra AGG?ACG?GAC?AAA?GGA?AAC?AGA?G 
PrimerPrimer sequence (5' ? 3')Sequence length (bp)
261–297-F* GCG?TCT?CTT?GTT?TCC?TGT?CCC 512 
261–297-R* GAC?TTA?CTT?CTT?GAT?GGC?AAA?CAC?AG 
467–1061-Fa TAC?GTG?GCT?TTC?CTG?AAG?CTG?T 789 
467–1061-Ra AGG?ACG?GAC?AAA?GGA?AAC?AGA?G 

*Sequencing region contains c.261delG and c.297A>G.

aSequencing region contains c.467C>T, c.646T>A, c.802G>A, c.930G>A, and c.1061delC.

Required amplification systems for sequencing included 5 µL of 10× Immobuffer, 2 µL of 50 mM of MgCl2, 2 µL of 2.5 mM of dNTPs, 1 µL of 5 U/µL of IMMOLASE DNA polymerase, 0.6 µL of each 10 µM of primer, deionized water to reach a total volume of 46 µL, and, finally, 4 µL of the DNA template (50–200 ng/µL). The PCR programs were as follows: predenaturation at 95°C for 10 min to activate the DNA polymerase; the amplification process consisted of 40 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 1 min. The final step was cooling at 40°C for 30 s.

PCR products were sent to Shanghai Sangon Biotech Co., Ltd., for sequencing. We then analyzed the results on Chromas Version 2.6.5. The Kappa test was used to analyze the consistency of the results between our established seven common ABO allele identification methods and the Sanger sequencing method.

The Results of Mimic Plasmids of Seven ABO Alleles’ Characteristic Loci and Sensitivity Test

Three loci of c.1061delC, c.297A, and c.646T>A of the FAM channel appeared in melting valleys at approximately 41°C, 51°C, and 63°C, respectively (Fig. 3a). The Tms corresponding to c.261delG, c.467C>T, c.802G>A, and c.930G>A of the HEX channel were approximately 45°C, 55°C, 62°C, and 66°C, respectively (Fig. 3b). The result of the sensitivity test showed that the detection limit of all loci was 1 × 102 copies/µL (Fig. 3).

Fig. 3.

2D PCR dual-channel detection of seven ABO allelic mutations in simulated plasmids and melting curves of the sensitivity test. In FAM and HEX channels, five diluted samples with concentrations from 1 × 106 to 1 × 102 copies/µL of mixed plasmids were selected. Deepened with the increase of the mixed plasmid concentration and a small shift of Tm occurred, especially in the low-temperature region.

Fig. 3.

2D PCR dual-channel detection of seven ABO allelic mutations in simulated plasmids and melting curves of the sensitivity test. In FAM and HEX channels, five diluted samples with concentrations from 1 × 106 to 1 × 102 copies/µL of mixed plasmids were selected. Deepened with the increase of the mixed plasmid concentration and a small shift of Tm occurred, especially in the low-temperature region.

Close modal

Mg2+ Concentration Refinement

The melting curves of the two channels were relatively better with Mg2+ concentration at 1.0 mM (dark purple curve), as shown in Figure 4.

Fig. 4.

Mg2+ concentration refinement test. The melting curve of the 1.0, 1.5, 2.0 and 2.5 mM for Mg2+ concentration were shown as dark purple, rose-red, dark blue, and light blue, respectively. When the Mg2+ concentration was 1.0 mM, the melting curve were relatively better. Meanwhile, Tm would shift to some extent with the increase of Mg2+ concentration.

Fig. 4.

Mg2+ concentration refinement test. The melting curve of the 1.0, 1.5, 2.0 and 2.5 mM for Mg2+ concentration were shown as dark purple, rose-red, dark blue, and light blue, respectively. When the Mg2+ concentration was 1.0 mM, the melting curve were relatively better. Meanwhile, Tm would shift to some extent with the increase of Mg2+ concentration.

Close modal

Identification of Seven Alleles of the ABO Blood Group

Table 4 showed the approximate Tms of c.1061delC, c.261G, c.297A, c.467C, and c.646T>A in the FAM channel as well as c.1061C, c.261delG, c.467C>T, c.802G>A, and c.930G>A in the HEX channel. The 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 each had four characteristic melting valleys.

Table 4.

Result judgments of the alleles

AlleleFAM channelHEX channel
c.1061delC (41°C)c.261G (45°C)c.297A (51°C)c.467C (56°C)c.646T>A (63°C)c.1061C (37°C)c.261delG (45°C)c.467C>T (55°C)c.802G>A (62°C)c.930G>A (66°C)
ABO*A1.01       
ABO*A1.02       
ABO*A2.01       
ABO*B.01       
ABO*O.01.01       
ABO*O.01.02       
ABO*O.02.01       
AlleleFAM channelHEX channel
c.1061delC (41°C)c.261G (45°C)c.297A (51°C)c.467C (56°C)c.646T>A (63°C)c.1061C (37°C)c.261delG (45°C)c.467C>T (55°C)c.802G>A (62°C)c.930G>A (66°C)
ABO*A1.01       
ABO*A1.02       
ABO*A2.01       
ABO*B.01       
ABO*O.01.01       
ABO*O.01.02       
ABO*O.02.01       

Clinical Sample Analysis

A total of 15 genotypes were detected in 260 clinical samples, including 6 genotypes in blood group A, 4 in blood group B, 3 in blood group O, and 2 in blood group AB. Among the 260 DNA samples, 259 were common genotypes, which were 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, and ABO*A1.02/B.01 in 1, 3, 1, 16, 19, 25, 18, 31, 15, 17, 30, 18, 6, and 59, respectively. One case with phenotype B, which is neither ABO*B.01/O.01.01 nor ABO*B.01/O.01.02 on genotyping because it does not contain c.297A (which is present in ABO*O.01.01) and c.646T>A (which is present in the ABO*O.01.02 allele). Compared to the ABO*A1.01 allele, this variant O allele contained c.106G>T, c.188G>A, c.189C>T, c.220C>T, c.261delG, and c.297A>G on exons 3–6 according to the results of Sanger sequencing. Referring to the ISBT allele table, this allele was tentatively designated as ABO*O.01. v (Tables 5,,6). Figure 5 shows the melting curves for the 15 genotypes.

Table 5.

Distribution of ABO genotypes in 260 samples

No.Blood groupGenotypeCasePercentage (%)*
ABO*A1.01/A1.02 1.54 
ABO*A1.01/O.01.01 4.62 
ABO*A1.01/O.01.02 1.54 
ABO*A1.02/A1.02 16 24.62 
ABO*A1.02/O.01.01 19 (1 case of Thai) 29.23 
ABO*A1.02/O.01.02 25 38.46 
ABO*B.01/B.01 18 27.69 
ABO*B.01/O.01.01 31 47.69 
ABO*B.01/O.01.02 15 23.08 
10 ABO*B.01/O.01.v 1.54 
11 ABO*O.01.01/O.01.01 17 26.15 
12 ABO*O.01.01/O.01.02 30 46.15 
13 ABO*O.01.02/O.01.02 18 27.69 
14 AB ABO*A1.01/B.01 9.23 
15 ABO*A1.02/B.01 59 90.77 
No.Blood groupGenotypeCasePercentage (%)*
ABO*A1.01/A1.02 1.54 
ABO*A1.01/O.01.01 4.62 
ABO*A1.01/O.01.02 1.54 
ABO*A1.02/A1.02 16 24.62 
ABO*A1.02/O.01.01 19 (1 case of Thai) 29.23 
ABO*A1.02/O.01.02 25 38.46 
ABO*B.01/B.01 18 27.69 
ABO*B.01/O.01.01 31 47.69 
ABO*B.01/O.01.02 15 23.08 
10 ABO*B.01/O.01.v 1.54 
11 ABO*O.01.01/O.01.01 17 26.15 
12 ABO*O.01.01/O.01.02 30 46.15 
13 ABO*O.01.02/O.01.02 18 27.69 
14 AB ABO*A1.01/B.01 9.23 
15 ABO*A1.02/B.01 59 90.77 

*The percentage (%) here refers to the percentage of each genotype in each blood group (65 cases of A, B, O, and AB, respectively), rounded off to two decimal places.

Table 6.

Results of 2D PCR and Sanger sequencing analysis

No.*FAM channelHEX channelGenotype2D PCR (N)aSanger sequencing (N)aAgreement (%)
c.1061delC (41°C)c.261G(45°C)c.297A(51°C)c.467C(56°C)c.646 T>A (63°C)c.1061C(37°C)c.261 delG (45°C)c.467 C>T (55°C)c.802G>A (62°C)c.930G>A (66°C)
     ABO*A1.01/A1.02 100 
     ABO*A1.01/O.01.01 100 
    ABO*A1.01/O.01.02 100 
      ABO*A1.02/A1.02 100 
    ABO*A1.02/O.01.01 13 13 100 
   ABO*A1.02/O.01.02 100 
      ABO*B.01/B.01 100 
    ABO*B.01/O.01.01 100 
    ABO*B.01/O.01.02 100 
10b      ABO*B.01/O.01.v 100 
11       ABO*O.01.01/O.01.01 10 10 100 
12      ABO*O.01.01/O.01.02 100 
13       ABO*O.01.02/O.01.02 100 
14      ABO*A1.01/B.01 100 
15     ABO*A1.02/B.01 100 
Sum    95 95 
No.*FAM channelHEX channelGenotype2D PCR (N)aSanger sequencing (N)aAgreement (%)
c.1061delC (41°C)c.261G(45°C)c.297A(51°C)c.467C(56°C)c.646 T>A (63°C)c.1061C(37°C)c.261 delG (45°C)c.467 C>T (55°C)c.802G>A (62°C)c.930G>A (66°C)
     ABO*A1.01/A1.02 100 
     ABO*A1.01/O.01.01 100 
    ABO*A1.01/O.01.02 100 
      ABO*A1.02/A1.02 100 
    ABO*A1.02/O.01.01 13 13 100 
   ABO*A1.02/O.01.02 100 
      ABO*B.01/B.01 100 
    ABO*B.01/O.01.01 100 
    ABO*B.01/O.01.02 100 
10b      ABO*B.01/O.01.v 100 
11       ABO*O.01.01/O.01.01 10 10 100 
12      ABO*O.01.01/O.01.02 100 
13       ABO*O.01.02/O.01.02 100 
14      ABO*A1.01/B.01 100 
15     ABO*A1.02/B.01 100 
Sum    95 95 

*A total of 15 genotypes were detected in 260 clinical samples.

aNumber of samples for agreement analysis of 2D PCR and Sanger sequencing.

bOne case with phenotype B, which is neither ABO*B.01/O.01.01 nor ABO*B.01/O.01.02 on genotyping because it does not contain c.297A (which is present in ABO*O.01.01), and c.646T>A (which is present in the ABO*O.01.02 allele).

Fig. 5.

Melting curves for the 15 genotypes. In FAM and HEX channels, the melting curves were displayed as blue and green, respectively. a Melting curves of genotypes in type A samples. b Melting curves of genotypes in type B samples. In the fourth genotype, the ABO*O allele is neither ABO*O.01.01 nor ABO*O.01.02 because it does not contain c.297A (which is present in ABO*O.01.01) and c.646T>A (which is present in the ABO*O.01.02 allele) in FAM channel, here named ABO*O.01.v. c Melting curves of genotypes in type O samples. d Melting curves of genotypes in type AB samples.

Fig. 5.

Melting curves for the 15 genotypes. In FAM and HEX channels, the melting curves were displayed as blue and green, respectively. a Melting curves of genotypes in type A samples. b Melting curves of genotypes in type B samples. In the fourth genotype, the ABO*O allele is neither ABO*O.01.01 nor ABO*O.01.02 because it does not contain c.297A (which is present in ABO*O.01.01) and c.646T>A (which is present in the ABO*O.01.02 allele) in FAM channel, here named ABO*O.01.v. c Melting curves of genotypes in type O samples. d Melting curves of genotypes in type AB samples.

Close modal

Methodological Agreement Analysis

The Kappa test showed completely consistent results for the two methods (Kappa = 1). Results of 2D PCR and Sanger sequencing analysis are presented in Table 6.

As early as 2000, Shea Ping Yip used single-tube single-lane multiplex PCR single-strand conformation polymorphism analysis to distinguish the seven common ABO alleles. The detection process comprised PCR amplification, SSCP analysis, and direct PCR product sequencing. Simply, SNPs are detected based on electrophoretic mobility differences of single-stranded DNA fragments with different sequences, and the addition of sequencing technology makes new allele identification easy [8]. However, the whole process is relatively time-consuming. ABO alleles could also be genotyped using PCR restriction fragment length polymorphism. But this approach requires enzyme digestion and electrophoresis in addition to PCR amplification [7, 22] and is easily affected by the effect of enzyme digestion. Certainly, PCR sequencing-based typing is the most accurate and reliable detection method that can detect new mutation points [9], but time and cost still remain the factors that need to be considered for large-scale clinical sample detection.

Compared with the disadvantages of the above methods, 2D PCR is more time-saving, and the whole process only needs PCR amplification, combined with the melting curves for the result analysis. This experiment can be completed in approximately 90 min, and finally, ABO genotypes can be distinguished according to the Tms. The most obvious advantage of this technology is its single-tube high-throughput detection, which can not only increase multiple detection channels but also improve the throughput of single channel. The previous article on the principle of 2D PCR technology mentioned that they produced a single probe that can identify at least five target genes and reduced the number of probes needed to detect multiple genes by introducing probe-targeting homologous tag reverse-complementary sequences into the products [13]. A probe can identify more target genes if we can avoid the problem of overlapping melting curves. This study detected ten characteristic loci of seven ABO alleles in a reaction tube by 2D PCR technology, and more target loci can be added if new ABO alleles are subsequently found. Meanwhile, it can complete all the detection processes without opening the tube after adding the samples. The closed-tube PCR method can largely avoid the potential laboratory pollution on the experimental results under the premise of standard operation. In addition, the study found a different rare ABO*O allele. The final identification depends on sequencing technology, but it shows that the technology can identify uncommon or rare ABO alleles at known mutation sites.

Detection of nine human papillomavirus subtypes and reference genes by 2D PCR has been previously reported [13]. In this study, we combined the PCR-SSP technique with 2D PCR. The mutant base was designed at the last position of the 3' end of the primer with tag, and the additional mismatch mutation was designed at the third–seventh position of the 3' end; thus, the specificity of the primers was improved. For another, the application of the hot-start enzyme avoided template mismatch at low temperature or primer dimer formation and greatly improved the specificity of amplification [23].

Some limitations remained despite the many abovementioned advantages. First, we observed that the melting curves of c.930G>A in the HEX channel were generally worse than those of other loci in combination with the sensitivity test of mixed plasmids. There were almost no melting curves, especially at the concentrations of ~102 and ~103 copies/µL. However, this does not affect the results of clinical samples because the concentration of extracted DNA from 200 µL blood samples is typical ~105 copies/µL. Second, judging the typing results is relatively difficult due to the seven ABO alleles producing more genotypes. The best way is to cooperate with relevant software companies to develop an ideal genotype interpretation plug-in to automatically read the PCR results to avoid errors and facilitate the rapid judgment of results. The study optimized the scheme of interpretation results with the help of Excel Office software (online suppl. Excel 1; for all online suppl. material, see www.karger.com/doi/10.1159/000530013). Third, primers can only be designed according to the known mutation sites, and new mutation sites cannot be identified. Finally, the clinical samples of individual alleles (ABO*A2.01, ABO*O.02.01) are difficult to obtain and can only be replaced by plasmids, but the complexity of clinical samples is much greater than that of plasmids, which is more convincing and can verify the accuracy of the methodology.

Establishing a new method for genotyping the ABO blood group system is greatly significant as the most important blood group system in transfusion medicine. Certainly, 2D PCR technology has great application space in the field of the molecular biology of other blood group systems, such as the RhD variants of the Rh blood group system and the human leukocyte antigen system. Briefly, the difference of antigens in a certain blood group system can be distinguished theoretically by 2D PCR as long as it is caused by SNP. Thus, the application of this technology in transfusion medicine will be more widely combined with the advantages of the methodology itself, worthy of further promotion and attention.

In summary, our study demonstrated that the 2D PCR technique could be used for molecular typing of the ABO blood group, which was efficient, rapid, accurate, and economical. Meanwhile, a rare ABO*O allele was found, indicating that the technology also could find uncommon or rare ABO alleles at known mutation sites.

Ethical approval was not required for this study in accordance with local guidelines. Written informed consent from participants was not required in accordance with local guidelines.

All the authors declare no conflict of interests.

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).

Guanghua Luo conceived and proposed the idea of 2D multiplex PCR. Guanghua Luo and Jin Chen designed all the experiments. Jin Chen, Yuxia Zhan, Jun Zhang, Yang Yu, and Shuang Yao performed the experiments and analyzed the data. Jin Chen wrote the manuscript, and Guanghua Luo revised and edited the manuscript. All the authors read and approved the final manuscript.

All the data reported in this paper are available in the manuscript or online supplementary information. For original sequencing results and melting curves of each sample, please contact 1041827254@qq.com.

1.
Yamamoto
F
,
Marken
J
,
Tsuji
T
,
White
T
,
Clausen
H
,
Hakomori
S
.
Cloning and characterization of DNA complementary to human UDP-GalNAc: fuc alpha 1----2Gal alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA
.
J Biol Chem
.
1990 Jan 15
265
2
1146
51
.
2.
Blumenfeld
OO
,
Patnaik
SK
.
Allelic genes of blood group antigens: a source of human mutations and cSNPs documented in the Blood Group Antigen Gene Mutation Database
.
Hum Mutat
.
2004 Jan
23
1
8
16
.
3.
Storry
JR
,
Olsson
ML
.
Genetic basis of blood group diversity
.
Br J Haematol
.
2004 Sep
126
6
759
71
.
4.
Daniels
G
.
The molecular genetics of blood group polymorphism
.
Transpl Immunol
.
2005 Aug
14
3–4
143
53
.
5.
Gassner
C
,
Schmarda
A
,
Nussbaumer
W
,
Schonitzer
D
.
ABO glycosyltransferase genotyping by polymerase chain reaction using sequence-specific primers
.
Blood
.
1996 Sep 1
88
5
1852
6
.
6.
Seltsam
A
,
Hallensleben
M
,
Kollmann
A
,
Burkhart
J
,
Blasczyk
R
.
Systematic analysis of the ABO gene diversity within exons 6 and 7 by PCR screening reveals new ABO alleles
.
Transfusion
.
2003 Apr
43
4
428
39
.
7.
Haak
W
,
Burger
J
,
Alt
KW
.
ABO genotyping by PCR-RFLP and cloning and sequencing
.
anthranz
.
2004 Dec
62
4
397
410
.
8.
Yip
SP
.
Single-tube multiplex PCR-SSCP analysis distinguishes 7 common ABO alleles and readily identifies new alleles
.
Blood
.
2000 Feb 15
95
4
1487
92
.
9.
Seltsam
A
,
Doescher
A
.
Sequence-based typing of human blood groups
.
Transfus Med Hemother
.
2009
;
36
(
3
):
204
12
.
10.
Muro
T
,
Fujihara
J
,
Imamura
S
,
Nakamura
H
,
Kimura-Kataoka
K
,
Toga
T
.
Determination of ABO genotypes by real-time PCR using allele-specific primers
.
Leg Med
.
2012 Jan
14
1
47
50
.
11.
Park
J-H
,
Han
J-H
,
Park
G
.
Rapid and reliable one-step ABO genotyping using direct real-time allele-specific PCR and melting curve analysis without DNA preparation
.
Indian J Hematol Blood Transfus
.
2019
;
35
(
3
):
531
7
.
12.
Soejima
M
,
Koda
Y
.
Detection of five common variants of ABO gene by a triplex probe-based fluorescence-melting-curve-analysis
.
Anal Biochem
.
2022 Jul 1
648
114668
.
13.
Zhan
Y
,
Zhang
J
,
Yao
S
,
Luo
G
.
High-throughput two-dimensional polymerase chain reaction technology
.
Anal Chem
.
2020 Jan 7
92
1
674
82
.
14.
Luo
G
,
Zheng
L
,
Zhang
X
,
Zhang
J
,
Nilsson-Ehle
P
,
Xu
N
.
Genotyping of single nucleotide polymorphisms using base-quenched probe: a method does not invariably depend on the deoxyguanosine nucleotide
.
Anal Biochem
.
2009 Mar 15
386
2
161
6
.
15.
Mao
H
,
Luo
G
,
Zhan
Y
,
Zhang
J
,
Yao
S
,
Yu
Y
.
The mechanism and regularity of quenching the effect of bases on fluorophores: the base-quenched probe method
.
Analyst
.
2018 Jul 9
143
14
3292
301
.
16.
Mao
H
,
Luo
G
,
Zhang
J
,
Xu
N
.
Detection of simultaneous multi-mutations using base-quenched probe
.
Anal Biochem
.
2018 Feb 15
543
79
81
.
17.
Chester
MA
,
Olsson
ML
.
The ABO blood group gene: a locus of considerable genetic diversity
.
Transfus Med Rev
.
2001 Jul
15
3
177
200
.
18.
Yip
SP
.
Sequence variation at the human ABO locus
.
Ann Hum Genet
.
2002 Jan
66
Pt 1
1
27
.
19.
Abegaz
SB
.
Human ABO blood groups and their associations with different diseases
.
Biomed Res Int
.
2021
;
2021
:
6629060
.
20.
Daniels
G
Human blood groups: introduction
Human Blood Groups
2013
.
21.
Thorisson
GA
,
Stein
LD
.
The SNP Consortium website: past, present and future
.
Nucleic Acids Res
.
2003 Jan 1
31
1
124
7
.
22.
Keller
JA
,
Horn
T
,
Scholz
S
,
Koenig
S
,
Keller
MA
.
Comparison of ABO genotyping methods: a study of two low-resolution polymerase chain reaction assays in a clinical testing laboratory
.
Immunohematology
.
2019 Dec
35
4
149
53
.
23.
Green
MR
,
Sambrook
J
.
Hot start polymerase chain reaction (PCR)
.
Cold Spring Harb Protoc
.
2018 May 1
2018
5
pdb.prot095125
.