Rapid and simple lateral flow detection for ADH1B and ALDH2 genotyping: PCR-restriction digestion-adapter ligation-lateral flow (PRALL) assay

IF 5.6 1区化学 Q1 CHEMISTRY, ANALYTICAL

Talanta Pub Date : 2025-07-15 DOI:10.1016/j.talanta.2025.128588

Naoto Shirasu, Shin'ichiro Yasunaga

{"title":"Rapid and simple lateral flow detection for ADH1B and ALDH2 genotyping: PCR-restriction digestion-adapter ligation-lateral flow (PRALL) assay","authors":"Naoto Shirasu, Shin'ichiro Yasunaga","doi":"10.1016/j.talanta.2025.128588","DOIUrl":null,"url":null,"abstract":"<div><div>Single nucleotide polymorphisms (SNPs) in <em>alcohol dehydrogenase 1B</em> (<em>ADH1B</em>) and <em>aldehyde dehydrogenase 2</em> (<em>ALDH2</em>) significantly impact ethanol metabolism and alcohol-related health risks. Rapid and simple genotyping methods for these genes are crucial for clinical diagnostics and point-of-care testing (POCT), but existing techniques often require complex procedures or specialized equipment. We developed and validated a novel genotyping method, termed a PCR-restriction digestion-adapter ligation-lateral flow (PRALL) assay. The PRALL assay integrates: (i) template preparation from buccal swab, (ii) duplex PCR amplification using biotinylated primers incorporating a <em>Bsl</em>I recognition site, (iii) simultaneous <em>Bsl</em>I restriction digestion and allele-specific ligation of hapten-labeled adapters in a single tube, and (iv) visual detection by lateral flow assay (LFA). The method was successfully validated using model templates and human genomic DNA. The complete PRALL assay, from crude lysate preparation to visual result, was performed in approximately 1 h. The assay demonstrated high sensitivity (limit of detection: ∼100 pg genomic DNA, corresponding to ∼15 allele copies) and achieved 100 % concordance with Sanger genotyping for both SNPs across 25 human samples. The unique integration of streamlined enzymatic steps with an instrument-free visual LFA readout overcomes key limitations of previous methods. This makes PRALL a promising and practical tool for facilitating personalized alcohol risk assessment and enhancing clinical diagnostics, demonstrating strong potential for deployment in various settings, including those closer to the point of care.</div></div>","PeriodicalId":435,"journal":{"name":"Talanta","volume":"297 ","pages":"Article 128588"},"PeriodicalIF":5.6000,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Talanta","FirstCategoryId":"92","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0039914025010781","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}

引用次数: 0

Abstract

Single nucleotide polymorphisms (SNPs) in alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) significantly impact ethanol metabolism and alcohol-related health risks. Rapid and simple genotyping methods for these genes are crucial for clinical diagnostics and point-of-care testing (POCT), but existing techniques often require complex procedures or specialized equipment. We developed and validated a novel genotyping method, termed a PCR-restriction digestion-adapter ligation-lateral flow (PRALL) assay. The PRALL assay integrates: (i) template preparation from buccal swab, (ii) duplex PCR amplification using biotinylated primers incorporating a BslI recognition site, (iii) simultaneous BslI restriction digestion and allele-specific ligation of hapten-labeled adapters in a single tube, and (iv) visual detection by lateral flow assay (LFA). The method was successfully validated using model templates and human genomic DNA. The complete PRALL assay, from crude lysate preparation to visual result, was performed in approximately 1 h. The assay demonstrated high sensitivity (limit of detection: ∼100 pg genomic DNA, corresponding to ∼15 allele copies) and achieved 100 % concordance with Sanger genotyping for both SNPs across 25 human samples. The unique integration of streamlined enzymatic steps with an instrument-free visual LFA readout overcomes key limitations of previous methods. This makes PRALL a promising and practical tool for facilitating personalized alcohol risk assessment and enhancing clinical diagnostics, demonstrating strong potential for deployment in various settings, including those closer to the point of care.

Abstract Image

查看原文本刊更多论文

快速和简单的侧流检测ADH1B和ALDH2基因分型：pcr -限制性消化-连接-侧流（PRALL）试验

乙醇脱氢酶1B （ADH1B）和醛脱氢酶2 （ALDH2）的单核苷酸多态性（snp）显著影响乙醇代谢和酒精相关的健康风险。这些基因的快速和简单的基因分型方法对于临床诊断和即时检测（POCT）至关重要，但现有技术往往需要复杂的程序或专门的设备。我们开发并验证了一种新的基因分型方法，称为pcr -限制性消化-适配器结扎-侧流（PRALL）测定。PRALL检测集成了：(i)从口腔拭子中制备模板，（ii）使用生物素化引物（包含BslI识别位点）进行双链PCR扩增，（iii）在单管中同时进行BslI限制性酶切和半抗原标记转接头的等位基因特异性连接，以及（iv）通过横向流动试验（LFA）进行视觉检测。使用模型模板和人类基因组DNA成功验证了该方法。从粗裂解液制备到目测结果，完整的PRALL分析大约在1小时内完成。该分析显示出高灵敏度（检测限：约100 pg基因组DNA，对应约15个等位基因拷贝），并且在25个人类样本中实现了与Sanger基因分型100%的一致性。独特的整合流线型酶的步骤与仪器无视觉LFA读出克服了以前的方法的关键限制。这使得PRALL成为促进个性化酒精风险评估和加强临床诊断的一个有希望和实用的工具，显示出在各种环境中部署的巨大潜力，包括那些更接近护理点的环境。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Talanta 化学-分析化学

CiteScore

12.30

自引率

4.90%

发文量

861

审稿时长

29 days

期刊介绍： Talanta provides a forum for the publication of original research papers, short communications, and critical reviews in all branches of pure and applied analytical chemistry. Papers are evaluated based on established guidelines, including the fundamental nature of the study, scientific novelty, substantial improvement or advantage over existing technology or methods, and demonstrated analytical applicability. Original research papers on fundamental studies, and on novel sensor and instrumentation developments, are encouraged. Novel or improved applications in areas such as clinical and biological chemistry, environmental analysis, geochemistry, materials science and engineering, and analytical platforms for omics development are welcome. Analytical performance of methods should be determined, including interference and matrix effects, and methods should be validated by comparison with a standard method, or analysis of a certified reference material. Simple spiking recoveries may not be sufficient. The developed method should especially comprise information on selectivity, sensitivity, detection limits, accuracy, and reliability. However, applying official validation or robustness studies to a routine method or technique does not necessarily constitute novelty. Proper statistical treatment of the data should be provided. Relevant literature should be cited, including related publications by the authors, and authors should discuss how their proposed methodology compares with previously reported methods.