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

IF 16.4 1区化学 Q1 CHEMISTRY, MULTIDISCIPLINARY

Accounts of Chemical Research Pub Date : 2023-04-24 eCollection Date: 2023-12-01 DOI:10.1159/000530013

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

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引用次数: 0

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.

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应用二维聚合酶链反应技术快速鉴定7个常见ABO等位基因

引言：人类ABO血型系统的分子生物学检测技术弥补了传统血清学分型技术在许多方面的局限性。本研究旨在建立一种新的方法，通过二维聚合酶链式反应（2D-PCR）鉴定7种常见的ABO等位基因（ABO*A1.01、ABO*A1.02、ABO*A2.01、ABO*B.01、ABO*O.01.01、ABO*O.01.02和ABO*O.02.01）。2D PCR可以通过用与荧光标记探针序列同源的标签标记特异性引物，在封闭的试管中识别多个靶基因，并在荧光探针与PCR特异性产物中的标签互补序列杂交后进行熔解曲线分析。本研究将2D PCR和PCR序列特异性引物（PCR-SSP）相结合，在单一反应中区分不同的等位基因，具有高通量的特点，并与其他分型技术进行了比较；可以在没有附加操作的情况下获得打字结果。方法：采用ABO*A1.01等位基因遗传序列作为参考序列。根据2D-PCR和PCR-SSP的原理，在外显子6和7上设计了7个常见ABO等位基因的特异性正义和反义引物。在FAM和HEX通道中分别检测到用于识别7个等位基因的单核苷酸多态性位点。采用2D-PCR对260份DNA样本进行快速ABO基因分型，其中95份样本进行Sanger测序。Kappa检验用于分析方法的一致性。结果：这7个等位基因在不同的单核苷酸多态性位点上各有4个特征性的熔解谷。共检测到15种基因型，包括ABO*A1.01/A1.02、ABO*A1.01/O.01.01、ABO*A.101/O.01.02、ABO*A102/A1.02、ABO*A1.02/O.01.01、ABO*A102/O.01.02和ABO*B.01/B.01、ABO*B.01/O.01.1、ABO*B.01/O.012、ABO*O.01.01/0.011、ABO*O.01.01/001.02、ABO*O.01.02/O.01.02，ABO*A1.01/B.01，ABO*A.02/B.01和ABO*B。01/O.01。v（根据测序结果，含有一个罕见的ABO*O等位基因）。Kappa检验显示2D PCR和Sanger测序的结果完全一致（Kappa=1）。结论：2D-PCR技术可用于ABO血型的分子分型，具有高效、快速、准确、经济的特点。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Accounts of Chemical Research 化学-化学综合

CiteScore

31.40

自引率

1.10%

发文量

312

审稿时长

2 months

期刊介绍： Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance. Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.