Rapid Identification of Seven Common <i>ABO</i> Alleles by Two-Dimensional Polymerase Chain Reaction Technology

Abstract

<b><i>Introduction:</i></b> The molecular biology detection technology of the human <i>ABO</i> 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 <i>ABO</i> alleles (<i>ABO</i>*<i>A1.01</i>, <i>ABO</i>*<i>A1.02</i>, <i>ABO</i>*<i>A2.01</i>, <i>ABO</i>*<i>B.01</i>, <i>ABO</i>*<i>O.01.01</i>, <i>ABO</i>*<i>O.01.02</i>, and <i>ABO</i>*<i>O.02.01</i>) 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. <b><i>Methods:</i></b> The <i>ABO</i>*<i>A1.01</i> allele genetic sequence was used as the reference sequence. The specific sense and antisense primers for seven common <i>ABO</i> 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 <i>ABO</i> genotyping by 2D PCR, and 95 of them were selected for Sanger sequencing. The <i>Kappa</i> test was used to analyze the agreement of the methodologies. <b><i>Results:</i></b> These 7 alleles each had four characteristic melting valleys at different single nucleotide polymorphism loci. A total of 15 genotypes were detected, including <i>ABO</i>*<i>A1.01/A1.02</i>, <i>ABO</i>*<i>A1.01/O.01.01</i>, <i>ABO</i>*<i>A1.01/O.01.02</i>, <i>ABO</i>*<i>A1.02/A1.02</i>, <i>ABO</i>*<i>A1.02/O.01.01</i>, <i>ABO</i>*<i>A1.02/O.01.02</i>, <i>ABO</i>*<i>B.01/B.01</i>, <i>ABO</i>*<i>B.01/O.01.01</i>, <i>ABO</i>*<i>B.01/O.01.02</i>, <i>ABO</i>*<i>O.01.01/O.01.01</i>, <i>ABO</i>*<i>O.01.01/O.01.02</i>, <i>ABO</i>*<i>O.01.02/O.01.02</i>, <i>ABO</i>*<i>A1.01/B.01</i>, <i>ABO</i>*<i>A1.02/B.01</i>, and <i>ABO</i>*<i>B.01/O.01. v</i> (containing a rare <i>ABO*O</i> allele, based on the sequencing results). The <i>Kappa</i> test showed completely consistent results for 2D PCR and Sanger sequencing (<i>Kappa</i> = 1). <b><i>Conclusion:</i></b> The 2D PCR technique could be used for molecular typing of the ABO blood group, which was efficient, rapid, accurate, and economical.