Sensitive and visual detection of SMA using RPA-Cas12a one-step assay with ssDNA-modified crRNA.

IF 2.9 3区 医学 Q2 MEDICAL LABORATORY TECHNOLOGY
Clinica Chimica Acta Pub Date : 2026-01-01 Epub Date: 2025-08-06 DOI:10.1016/j.cca.2025.120537
Qinyi Huang, Changzhi Xu, Shiqi Liu, Haihong Shi, XiaoBin Zhong, Shumin Zhao, Fengxiang Wei, Lvyuan Fan, Cui Wang, Yuanqing Li, Jia Tang
{"title":"Sensitive and visual detection of SMA using RPA-Cas12a one-step assay with ssDNA-modified crRNA.","authors":"Qinyi Huang, Changzhi Xu, Shiqi Liu, Haihong Shi, XiaoBin Zhong, Shumin Zhao, Fengxiang Wei, Lvyuan Fan, Cui Wang, Yuanqing Li, Jia Tang","doi":"10.1016/j.cca.2025.120537","DOIUrl":null,"url":null,"abstract":"<p><p>The irreversibility and lethality of Spinal Muscular Atrophy (SMA) underscore the urgency of newborn screening, as diagnostic delay in neonates causes irreversible motor neuron degeneration and poor outcomes. Current SMA detection methods are hindered by high costs, dependence on specialized equipment, and technical complexity, restricting their implementation in primary care setting. Here, we proposed a fast and sensitive SMA-(Recombinase Polymerase Amplification) RPA-Cas12a detection assay based on suboptimal protospacer adjacent motif (sPAM) and 3'-end ssDNA-modified crRNA, named SPSMC. The crRNA is designed based on the sPAM to enhance the specificity of SMN1 gene detection. The competition between RPA and Cas12a digestion for target DNA was resolved by using 3'-end ssDNA-modified crRNA. With ALB as a reference gene, this method can detect DNA at concentrations as low as 1.8 pM within 1 h. The sensitivity and specificity of the proposed method in differentiating SMA patients from non-SMA individuals were both 100 %. This strategy has been used for the detection of the SMN1 gene, which saves time, reduces contamination risks, and offers new possibilities for future point-of-care screening of SMA. In addition, the SPSMC system was successfully adapted to SMA lateral flow assay format and validated using 66 clinical samples, demonstrating 100 % sensitivity and specificity. The method is straightforward to perform, requires no bulky equipment, maintains full portability, and is more suitable for large-scale neonatal screening scenarios compared with traditional methods.</p>","PeriodicalId":10205,"journal":{"name":"Clinica Chimica Acta","volume":" ","pages":"120537"},"PeriodicalIF":2.9000,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinica Chimica Acta","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.cca.2025.120537","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/8/6 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"MEDICAL LABORATORY TECHNOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

The irreversibility and lethality of Spinal Muscular Atrophy (SMA) underscore the urgency of newborn screening, as diagnostic delay in neonates causes irreversible motor neuron degeneration and poor outcomes. Current SMA detection methods are hindered by high costs, dependence on specialized equipment, and technical complexity, restricting their implementation in primary care setting. Here, we proposed a fast and sensitive SMA-(Recombinase Polymerase Amplification) RPA-Cas12a detection assay based on suboptimal protospacer adjacent motif (sPAM) and 3'-end ssDNA-modified crRNA, named SPSMC. The crRNA is designed based on the sPAM to enhance the specificity of SMN1 gene detection. The competition between RPA and Cas12a digestion for target DNA was resolved by using 3'-end ssDNA-modified crRNA. With ALB as a reference gene, this method can detect DNA at concentrations as low as 1.8 pM within 1 h. The sensitivity and specificity of the proposed method in differentiating SMA patients from non-SMA individuals were both 100 %. This strategy has been used for the detection of the SMN1 gene, which saves time, reduces contamination risks, and offers new possibilities for future point-of-care screening of SMA. In addition, the SPSMC system was successfully adapted to SMA lateral flow assay format and validated using 66 clinical samples, demonstrating 100 % sensitivity and specificity. The method is straightforward to perform, requires no bulky equipment, maintains full portability, and is more suitable for large-scale neonatal screening scenarios compared with traditional methods.

利用RPA-Cas12a一步法和ssdna修饰的crRNA对SMA进行灵敏和视觉检测。
脊髓性肌萎缩症(SMA)的不可逆性和致死率强调了新生儿筛查的紧迫性,因为新生儿的诊断延迟会导致不可逆的运动神经元变性和不良预后。目前的SMA检测方法受到高成本、依赖专业设备和技术复杂性的阻碍,限制了它们在初级保健环境中的实施。本研究基于次优原间隔邻近基序(suboptimal protospacer邻基序,sPAM)和3'端ssdna修饰的crRNA (SPSMC),提出了一种快速灵敏的SMA-(Recombinase Polymerase Amplification,重组酶聚合酶扩增)RPA-Cas12a检测方法。crRNA是在sPAM的基础上设计的,目的是提高SMN1基因检测的特异性。利用3'端ssdna修饰的crRNA解决了RPA和Cas12a酶切靶DNA的竞争。该方法以ALB为内参基因,可在1 h内检测浓度低至1.8 pM的DNA。该方法鉴别SMA患者与非SMA个体的敏感性和特异性均为100% %。该策略已用于SMN1基因的检测,从而节省了时间,降低了污染风险,并为未来的SMA即时筛查提供了新的可能性。此外,SPSMC系统已成功适应SMA横向流动分析格式,并使用66个临床样本进行验证,显示出100% %的灵敏度和特异性。该方法操作简单,不需要笨重的设备,保持充分的便携性,与传统方法相比,更适合大规模的新生儿筛查场景。
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
Clinica Chimica Acta
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.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信