{"title":"基于CRISPR/Cas12a和DNAzyme的鼠疫耶尔森氏菌elisa样灵敏视觉检测系统","authors":"Yingqing Mao, Ruichen Lv, Hao Shao, Yong Zhao, Junhu Wang, Qiong Chen, Haiming Yi, Yixin Ge, Hongming Wang, Yuexi Li, Yong Qi","doi":"10.1128/jcm.00274-25","DOIUrl":null,"url":null,"abstract":"<p><p><i>Yersinia pestis</i> 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 <i>Y. pestis</i>. A novel specific gene sequence (CH57_3927) was screened for the detection target of <i>Y. pestis</i>. 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<sup>2-</sup>) 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 <i>Y. pestis</i> from all other closely related species, with a detection limit of 1 copy/reaction. Evaluated using <i>Y. pestis</i> 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 <i>Yersinia pestis</i> 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 <i>Y. pestis</i> outbreaks, providing effective support for plague detection, prevention, and control within primary medical and health institutions.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0027425"},"PeriodicalIF":5.4000,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345247/pdf/","citationCount":"0","resultStr":"{\"title\":\"An ELISA-like sensitive and visual detection system targeting <i>Yersinia pestis</i> based on CRISPR/Cas12a and DNAzyme.\",\"authors\":\"Yingqing Mao, Ruichen Lv, Hao Shao, Yong Zhao, Junhu Wang, Qiong Chen, Haiming Yi, Yixin Ge, Hongming Wang, Yuexi Li, Yong Qi\",\"doi\":\"10.1128/jcm.00274-25\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><i>Yersinia pestis</i> 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 <i>Y. pestis</i>. A novel specific gene sequence (CH57_3927) was screened for the detection target of <i>Y. pestis</i>. 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<sup>2-</sup>) 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 <i>Y. pestis</i> from all other closely related species, with a detection limit of 1 copy/reaction. Evaluated using <i>Y. pestis</i> 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 <i>Yersinia pestis</i> 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 <i>Y. pestis</i> outbreaks, providing effective support for plague detection, prevention, and control within primary medical and health institutions.</p>\",\"PeriodicalId\":15511,\"journal\":{\"name\":\"Journal of Clinical Microbiology\",\"volume\":\" \",\"pages\":\"e0027425\"},\"PeriodicalIF\":5.4000,\"publicationDate\":\"2025-08-13\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345247/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Clinical Microbiology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1128/jcm.00274-25\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/7/24 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q1\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Clinical Microbiology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1128/jcm.00274-25","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/7/24 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
An ELISA-like sensitive and visual detection system targeting Yersinia pestis based on CRISPR/Cas12a and DNAzyme.
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.
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.