Detection of five common variants of ABO gene by a triplex probe-based fluorescence-melting-curve-analysis

Soejima, Mikiko; Koda, Yoshiro

doi:10.1016/j.ab.2022.114668

Cited by 1 publication

(2 citation statements)

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“…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][3][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][11][12]. Various methodologies have both advantages and disadvantages.…”

Section: Introductionmentioning

confidence: 99%

“…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 timeconsuming 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][15][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.…”

Section: Introductionmentioning

confidence: 99%

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

Chen

Zhan

Zhang

et al. 2023

Transfus Med Hemother

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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.

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