Mao Yingqing, Lv Ruichen, Shao Hao, Zhao Yong, Wang Junhu, Chen Qiong, Yi Haiming, Ge Yixin, Wang Hongming, Li Yuexi, Qi Yong
Huadong Research Institute for Medicine and Biotechniques, Nanjing, China.
Teachers College, Columbia University, Manhattan, New York, USA.
J Clin Microbiol. 2025 Aug 13;63(8):e0027425. doi: 10.1128/jcm.00274-25. Epub 2025 Jul 24.
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 . A novel specific gene sequence (CH57_3927) was screened for the detection target of . 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) (ABTS) 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 from all other closely related species, with a detection limit of 1 copy/reaction. Evaluated using 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 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 outbreaks, providing effective support for plague detection, prevention, and control within primary medical and health institutions.
是鼠疫的病原体，鼠疫是一种对人类有潜在毁灭性后果的疾病。在此，我们基于成簇规律间隔短回文重复序列（CRISPR）检测和脱氧核酶开发了一种类似酶联免疫吸附测定的视觉检测方法，用于对进行经济高效且高灵敏度的检测。筛选了一种新的特异性基因序列（CH57_3927）作为的检测靶点。分别建立并优化了重组酶辅助扩增（RAA）测定、CRISPR/Cas12a检测测定和基于G-四链体（G4）脱氧核酶的显色测定。通过进一步优化其组分以提高它们之间的兼容性，将这三种优化后的测定整合到一种先进的类似酶联免疫吸附测定的视觉检测方法——RAA-CRISPR/Cas12a-脱氧核酶（RCCD）中。扩增的靶序列与crRNA结合并激活Cas12a核酸酶以反式切割G4。结果，切割后的G4无法与血红素结合以发挥过氧化物酶活性，从而阻碍了2,2'-联氮-双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）比色反应的催化。因此，阴性样品呈现深绿色，而阳性产物几乎无色，便于肉眼进行视觉区分。此外，RCCD检测平台能够有效区分与所有其他密切相关的物种，检测限为1拷贝/反应。使用掺入DNA的血样和未感染样品进行评估，灵敏度和特异性均为100%。该方法在检测临床样品中的靶点方面显示出巨大潜力，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。重要性我们利用Mauve软件筛选特异性基因，并整合CRISPR-Cas12a、RAA扩增和G-四链体脱氧核酶技术，建立了一种先进的类似酶联免疫吸附测定的视觉检测方法。与依赖荧光标记的单链DNA报告分子或侧向流动（LF）试纸条的传统CRISPR检测方法相比，这种视觉检测方法提供了一种更具成本效益的替代方案。仅需一台恒温设备，它提高了快速现场筛查疫情的便利性，为基层医疗卫生机构内的鼠疫检测、预防和控制提供了有效支持。
