An ELISA-like sensitive and visual detection system targeting Yersinia pestis based on CRISPR/Cas12a and DNAzyme.

IF 5.4 2区 医学 Q1 MICROBIOLOGY
Journal of Clinical Microbiology Pub Date : 2025-08-13 Epub Date: 2025-07-24 DOI:10.1128/jcm.00274-25
Yingqing Mao, Ruichen Lv, Hao Shao, Yong Zhao, Junhu Wang, Qiong Chen, Haiming Yi, Yixin Ge, Hongming Wang, Yuexi Li, Yong Qi
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引用次数: 0

Abstract

Yersinia pestis is the causative agent of plague, a human disease with potentially devastating consequences. Here, we developed an enzyme-linked immunosorbent assay-like visual detection method based on clustered regularly interspaced short palindromic repeats (CRISPR) detection and DNAzyme for the cost-effective and highly sensitive detection of Y. pestis. A novel specific gene sequence (CH57_3927) was screened for the detection target of Y. pestis. The recombinase-aided amplification (RAA) assay, CRISPR/Cas12a detection assay, and G-quadruplex (G4) DNAzyme-based color development assay were separately established and optimized. These three optimized assays were integrated into an advanced ELISA-like visual detection method-RAA-CRISPR/Cas12a-DNAzyme (RCCD)-by further optimization of their components to improve the compatibility between them. The amplified target sequence binds to crRNA and activates the Cas12a nucleases for trans-cleave G4. As a result, the cleaved G4 is unable to bind with hemin to exert peroxidase activity, thus impeding the catalysis of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2-) colorimetric reaction. Consequently, negative samples exhibit a dark green coloration, while the positive products appear nearly colorless, facilitating visual differentiation with the naked eye. In addition, the RCCD detection platform effectively distinguished Y. pestis from all other closely related species, with a detection limit of 1 copy/reaction. Evaluated using Y. pestis DNA-spiked blood samples and uninfected samples, both sensitivity and specificity were 100%. The method shows significant potential for detecting targets in clinical samples and is well-suited for use in resource-limited environments. It offers advantages such as visual detection, batch detection, and low cost.IMPORTANCEWe utilized Mauve software to screen Yersinia pestis specific genes and integrated CRISPR-Cas12a, RAA amplification, and G-quadruplex DNAzyme technology to establish an advanced ELISA-like visual detection method. The visual detection method offers a more cost-effective alternative compared to the conventional CRISPR detection method that relies on fluorescence-labeled ssDNA reporter or lateral flow (LF) test strips. With only one thermostatic device required, it enhances the convenience of rapid on-site screening of Y. pestis outbreaks, providing effective support for plague detection, prevention, and control within primary medical and health institutions.

基于CRISPR/Cas12a和DNAzyme的鼠疫耶尔森氏菌elisa样灵敏视觉检测系统
鼠疫耶尔森菌是鼠疫的病原体,鼠疫是一种具有潜在破坏性后果的人类疾病。在这里,我们开发了一种基于聚类规则间隔短回文重复(CRISPR)检测和DNAzyme的酶联免疫吸附法样视觉检测方法,用于成本效益高、灵敏度高的鼠疫菌检测。筛选到一个新的特异性基因序列CH57_3927作为鼠疫菌的检测靶点。分别建立并优化了重组酶辅助扩增(RAA)法、CRISPR/Cas12a检测法和g -四重体(G4) dnazyme显色法。这三种优化的检测方法通过进一步优化其组分,以提高它们之间的相容性,整合到一种先进的类似elisa的视觉检测方法- raa - crispr /Cas12a-DNAzyme (RCCD)中。扩增后的靶序列与crRNA结合,激活Cas12a核酸酶进行反式裂解G4。因此,裂解后的G4不能与hemin结合发挥过氧化物酶活性,从而阻碍了2,2'-氮基-双(3-乙基苯并噻唑-6-磺酸)(ABTS2-)比色反应的催化作用。因此,阴性样品呈现深绿色,而阳性产品几乎无色,便于肉眼视觉区分。此外,RCCD检测平台可有效区分鼠疫杆菌与其他近缘种,检测限为1拷贝/反应。使用含有鼠疫杆菌dna的血液样本和未感染样本进行评估,敏感性和特异性均为100%。该方法显示了在临床样品中检测目标的巨大潜力,并且非常适合在资源有限的环境中使用。它具有视觉检测、批量检测和低成本等优点。我们利用Mauve软件筛选鼠疫耶尔森菌特异性基因,结合CRISPR-Cas12a、RAA扩增、g -四重体DNAzyme技术,建立了一种先进的类似elisa的视觉检测方法。与依赖荧光标记的ssDNA报告基因或侧流(LF)试纸条的传统CRISPR检测方法相比,视觉检测方法提供了一种更具成本效益的替代方法。仅需要一台恒温装置,提高了鼠疫菌现场快速筛查的便利性,为基层医疗卫生机构鼠疫检测、预防和控制提供了有效支持。
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
Journal of Clinical Microbiology
Journal of Clinical Microbiology 医学-微生物学
CiteScore
17.10
自引率
4.30%
发文量
347
审稿时长
3 months
期刊介绍: The Journal of Clinical Microbiology® disseminates the latest research concerning the laboratory diagnosis of human and animal infections, along with the laboratory's role in epidemiology and the management of infectious diseases.
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