One-tube genotyping using TaqMan-PCR without DNA extraction.

IF 1.6 4区医学 Q3 HEMATOLOGY

Vox Sanguinis Pub Date : 2025-08-01 Epub Date: 2025-05-21 DOI:10.1111/vox.70054

Toru Miyagi, Ayano Tahara, Hironori Kobayashi, Nelson H Tsuno, Kazuo Muroi

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引用次数: 0

Abstract

Background and objectives: Genotyping has become an indispensable technique widely used in transfusion medicine. However, further simplification of the process is essential. Among the simplest methods, TaqMan-PCR is prominent. Nevertheless, challenges related to time, cost and the risk of sample mix-up during the DNA extraction process persist. To address these challenges, we have developed a novel blood processing method.

Materials and methods: Whole blood collected from healthy adult blood donors was heated at 95°C for 5 min and then used as a TaqMan-PCR template. The human platelet antigen (HPA)-1, -2, -3, -4, -5 and -15 genotypes of 60 individuals were evaluated.

Results: The results were fully consistent with those obtained through routine testing using PCR with reverse-sequence-specific oligonucleotide (rSSO) hybridization, confirming the reliability of our approach.

Conclusion: The developed 'one-tube method' significantly reduces the risk of sample mix-up while offering considerable time and cost savings. Furthermore, this method has broader applicability to other genes, as the polymorphic regions tested in this study exhibited variability in guanine-cytosine content and amplicon lengths.

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TaqMan-PCR单管基因分型，无需提取DNA。

背景与目的：基因分型已成为输血医学中广泛应用的一项不可或缺的技术。然而，进一步简化这一过程是必要的。在最简单的方法中，TaqMan-PCR最为突出。然而，在DNA提取过程中，与时间、成本和样品混淆风险相关的挑战仍然存在。为了应对这些挑战，我们开发了一种新的血液处理方法。材料与方法：采集健康成人献血者全血，95℃加热5 min，作为TaqMan-PCR模板。对60例人血小板抗原(HPA)-1、-2、-3、-4、-5和-15基因型进行检测。结果：结果与常规PCR与反序列特异性寡核苷酸（reverse-sequence-specific oligonucleotide, rSSO）杂交检测结果完全一致，证实了方法的可靠性。结论：所开发的“单管法”显著降低了样品混淆的风险，同时节省了大量的时间和成本。此外，该方法对其他基因具有更广泛的适用性，因为本研究中测试的多态性区域在鸟嘌呤-胞嘧啶含量和扩增子长度方面表现出可变性。

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

Vox Sanguinis 医学-血液学

CiteScore

4.40

自引率

11.10%

发文量

156

审稿时长

6-12 weeks

期刊介绍： Vox Sanguinis reports on important, novel developments in transfusion medicine. Original papers, reviews and international fora are published on all aspects of blood transfusion and tissue transplantation, comprising five main sections: 1) Transfusion - Transmitted Disease and its Prevention: Identification and epidemiology of infectious agents transmissible by blood; Bacterial contamination of blood components; Donor recruitment and selection methods; Pathogen inactivation. 2) Blood Component Collection and Production: Blood collection methods and devices (including apheresis); Plasma fractionation techniques and plasma derivatives; Preparation of labile blood components; Inventory management; Hematopoietic progenitor cell collection and storage; Collection and storage of tissues; Quality management and good manufacturing practice; Automation and information technology. 3) Transfusion Medicine and New Therapies: Transfusion thresholds and audits; Haemovigilance; Clinical trials regarding appropriate haemotherapy; Non-infectious adverse affects of transfusion; Therapeutic apheresis; Support of transplant patients; Gene therapy and immunotherapy. 4) Immunohaematology and Immunogenetics: Autoimmunity in haematology; Alloimmunity of blood; Pre-transfusion testing; Immunodiagnostics; Immunobiology; Complement in immunohaematology; Blood typing reagents; Genetic markers of blood cells and serum proteins: polymorphisms and function; Genetic markers and disease; Parentage testing and forensic immunohaematology. 5) Cellular Therapy: Cell-based therapies; Stem cell sources; Stem cell processing and storage; Stem cell products; Stem cell plasticity; Regenerative medicine with cells; Cellular immunotherapy; Molecular therapy; Gene therapy.