利用重组酶聚合酶扩增结合横向流动条带快速可靠地检测G6PD突变

IF 3.2 3区医学 Q2 MEDICAL LABORATORY TECHNOLOGY

Clinica Chimica Acta Pub Date : 2025-05-03 DOI:10.1016/j.cca.2025.120345

Beatriz Aira C. Jacob , Warangkhana Songsungthong , Ubolsree Leartsakulpanich , Usa Boonyuen

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引用次数: 0

摘要

葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症是最常见的酶病，全球约有5亿人受到影响。它源于G6PD基因的遗传突变，导致对药物性溶血性贫血和新生儿严重黄疸的易感性增加。虽然表型检测是常用的，但基因检测在准确诊断G6PD缺乏症方面的价值越来越得到认可，特别是在杂合子女性和新生儿中。本研究旨在开发和评估一种快速、可现场部署的基因检测方法，用于检测泰国四种常见的G6PD变异：G6PD高河(c.95A >；G)， G6PD玛希隆(c.487G >；A), G6PD Viangchan (c.871G >；A)和G6PD Canton (c.1376G >；T)。该检测利用等位基因特异性引物（包含锁定核酸）进行重组酶聚合酶扩增以增强特异性，然后采用横向流动条带检测以获得视觉读数。在37˚C下，检测可在45分钟内得出结果。单路检测具有100%的诊断敏感性（置信区间（CI）： 95.01 - 100.0%）和特异性（CI: 95.49 - 100.0%）。双联检测（高河+广州和玛希多+ Viangchan）也显示出100%的诊断敏感性（CI: 94.87 - 100.0%）和特异性（CI: 91.19 - 100.0%）。高河、玛希多、伟江和广州的检测限分别为0.25、1.00、0.50和0.50 ng/µL。双相检测结果显示，玛希多+强参的lod为0.10 ng/μL，高河+广的lod为10.00 ng/μL。突变型、野生型和非目标等位基因之间的波段强度差异为5.25 ~ 19.61像素，可以进行明显的等位基因区分。这种创新的诊断工具为即时基因检测提供了一种快速、可靠和可获得的解决方案，有可能改善G6PD缺乏症高负担地区的临床管理和医疗保健结果。

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Rapid and reliable detection of G6PD mutations using recombinase polymerase amplification coupled with lateral flow strip

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy, affecting approximately 500 million people worldwide. It results from inherited mutations in the G6PD gene, causing increased susceptibility to drug-induced hemolytic anemia and severe neonatal jaundice. While phenotypic tests are commonly used, genetic testing is increasingly recognized for its value in the accurate diagnosis of G6PD deficiency, especially in heterozygous females and newborns.

This study aimed to develop and evaluate a rapid, field-deployable genetic test for the detection of four common G6PD variants in Thailand: G6PD Gaohe (c.95A > G), G6PD Mahidol (c.487G > A), G6PD Viangchan (c.871G > A), and G6PD Canton (c.1376G > T). The assays utilize recombinase polymerase amplification with allele-specific primers incorporating locked nucleic acids to enhance specificity, followed by lateral flow strip detection for visual readout.

The assays deliver results within 45 min at 37 ˚C. Singleplex detection demonstrated 100 % diagnostic sensitivity (Confidence interval (CI): 95.01–100.0 %) and specificity (CI: 95.49–100.0 %). Duplex assays (Gaohe + Canton and Mahidol + Viangchan) also demonstrated 100 % diagnostic sensitivity (CI: 94.87–100.0 %) and specificity (CI: 91.19–100.0 %). Limits of detection (LOD) for singleplex assays were 0.25, 1.00, 0.50, and 0.50 ng/µL, for Gaohe, Mahidol, Viangchan, and Canton, respectively. Duplex assays showed LODs of 0.10 ng/μL for Mahidol + Viangchan and 10.00 ng/μL for Gaohe + Canton. Band intensity differences ranged from 5.25 to 19.61 pixels between mutant, wild-type, and nontarget alleles, enabling clear allele discrimination.

This innovative diagnostic tool offers a rapid, reliable, and accessible solution for point-of-care genetic testing, with the potential to improve clinical management and healthcare outcomes in regions with a high burden of G6PD deficiency.

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来源期刊

Clinica Chimica Acta 医学-医学实验技术

CiteScore

10.10

自引率

2.00%

发文量

1268

审稿时长

23 days

期刊介绍： The Official Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Clinica Chimica Acta is a high-quality journal which publishes original Research Communications in the field of clinical chemistry and laboratory medicine, defined as the diagnostic application of chemistry, biochemistry, immunochemistry, biochemical aspects of hematology, toxicology, and molecular biology to the study of human disease in body fluids and cells. The objective of the journal is to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management. Reports of an applied clinical character are also welcome. Papers concerned with normal metabolic processes or with constituents of normal cells or body fluids, such as reports of experimental or clinical studies in animals, are only considered when they are clearly and directly relevant to human disease. Evaluation of commercial products have a low priority for publication, unless they are novel or represent a technological breakthrough. Studies dealing with effects of drugs and natural products and studies dealing with the redox status in various diseases are not within the journal''s scope. Development and evaluation of novel analytical methodologies where applicable to diagnostic clinical chemistry and laboratory medicine, including point-of-care testing, and topics on laboratory management and informatics will also be considered. Studies focused on emerging diagnostic technologies and (big) data analysis procedures including digitalization, mobile Health, and artificial Intelligence applied to Laboratory Medicine are also of interest.