聚类规则间隔短回文重复序列/ crispr相关蛋白和Argonaute核酸酶的病毒检测

IF 2.9 3区医学 Q2 MEDICAL LABORATORY TECHNOLOGY

Clinica Chimica Acta Pub Date : 2026-01-01 Epub Date: 2025-08-07 DOI:10.1016/j.cca.2025.120526

Liang Xu, Xuping Wu

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引用次数: 0

摘要

以2019年新冠肺炎疫情为例，病毒大流行对人类健康和社会稳定构成严重威胁。传统的病毒检测方法，如聚合酶链反应（PCR）通常需要训练有素的人员，昂贵的设备和2-4 小时的处理时间。基于集群规则间隔短回文量重复序列(CRISPR)/CRISPR相关蛋白（Cas）和Argonaute （Ago）系统的检测方法实现了原子摩尔灵敏度或单拷贝检测限，单碱基特异性在1 h内，不需要复杂或昂贵的仪器。本文首先介绍了CRISPR/Cas系统（Cas9、Cas12、Cas13）和Ago系统的机制和功能。它还介绍了具有重大社会影响的病毒，并继续审查这些系统在单一和多重病毒检测中的应用。单病毒检测包括最近开发的DNA/ rna激活Cas9检测（daca /RACD），利用Cas9反式裂解活性，基于cas12的DNA内切酶靶向CRISPR反式报告器（DETECTR）具有原子摩尔灵敏度，基于CRISPR/ cas13的荧光纳米颗粒SARS-CoV-2 （CFNS）具有1 拷贝/mL的灵敏度，量子点报告器，无扩增的手机检测，检测31 拷贝/μL，无需扩增。多重病毒检测包括集成CRISPR/Cas12a和多重重组酶聚合酶扩增（MiCaR）的微流控装置，通过微流控芯片具有空间辨别功能，实现30-plex检测；pago介导的核酸检测（PAND）利用ago产生的引导序列进行5-plex检测；特异性高灵敏度酶报告解锁v2 （SHERLOCKv2）利用多酶单反应系统实现4-plex检测；以及支持100多种靶标检测的核酸多路评价（CARMEN）。最后，本文讨论了CRISPR/Cas和基于ago的检测方法面临的挑战，包括Cas9/12对原间隔器邻近Motif （PAM）序列的要求，核酸提取/扩增导致的反应时间延长，以及核酸酶和crrna等核心组分的不稳定性。检测特异性和多路性有待进一步提高。未来的发展方向是提高检测特异性，发展多重功能和推进POCT。使用CRISPR/Cas和Ago系统开发诊断工具可以改变分子诊断，这些工具有望在全球范围内轻松获得。它们对于精确识别和战略性控制传染病传播至关重要。

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Viral detection using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein and Argonaute nucleases.

Viral pandemics pose severe threats to human health and societal stability, exemplified by the COVID-19 outbreak in 2019. Conventional viral detection methods such as Polymerase chain reaction (PCR) typically require trained personnel, expensive equipment, and 2-4 h for processing. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) system-based detection methods achieve attomolar sensitivity or single-copy detection limits with single-base specificity within 1 h, without requiring complex or costly instruments. This review firstly introduces the mechanisms and functions of CRISPR/Cas systems (Cas9, Cas12, Cas13) and Ago systems. It also introduces viruses with significant social impact, and continued with reviewing applications of these systems in single and multiplex virus detection. Single viral detection includes recently developed DNA/RNA-activated Cas9 detection (DACD/RACD) using Cas9 trans-cleavage activity, Cas12-based DNA Endonuclease-targeted CRISPR Trans Reporter (DETECTR) with attomolar sensitivity, CRISPR/Cas13a-based Fluorescent Nanoparticle SARS-CoV-2 (CFNS) achieving 1 copy/mL sensitivity with quantum dot reporters, and amplification-free mobile phone detection detecting 31 copies/μL without amplification. Multiplex viral detection includes Microfluidic Device Integrated with CRISPR/Cas12a and Multiplex Recombinase Polymerase Amplification (MiCaR) enabling 30-plex detection through microfluidic chips with spatial discrimination, PfAgo-mediated Nucleic acid Detection (PAND) utilizing Ago-produced guide sequences for 5-plex detection, Specific High-Sensitivity Enzymatic Reporter UnLOCKing v2 (SHERLOCKv2) achieving 4-plex detection with multi-enzyme single-reaction systems, and Multiplexed Evaluation of Nucleic acids (CARMEN) supporting over 100 target assays. Finally, this review discusses challenges in CRISPR/Cas and Ago-based detection methods, including Protospacer Adjacent Motif (PAM) sequence requirements for Cas9/12, prolonged reaction times due to nucleic acid extraction/amplification, and instability of core components like nucleases and crRNAs. Detection specificity and multiplex capabilities could be further improved. Future directions are outlined for improving detection specificity, developing multiplex capabilities and advancing POCT. Developing diagnostic tools using CRISPR/Cas and Ago systems could transform molecular diagnostics, such tools promise to be easily accessible worldwide. They are essential for precise identification and strategic containment of infectious disease transmission.

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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.