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基于 ERA-CRISPR/Cas12a 的快速、特异诊断检测方法用于 。

ERA-CRISPR/Cas12a-based, fast and specific diagnostic detection for .

机构信息

The Institute of Pathogenic Biology, Hengyang Medical School, University of South China, Hengyang, China.

Department of Clinical Laboratory, The Second People's Hospital of Foshan, Foshan, China.

出版信息

Front Cell Infect Microbiol. 2024 Nov 1;14:1477422. doi: 10.3389/fcimb.2024.1477422. eCollection 2024.


DOI:10.3389/fcimb.2024.1477422
PMID:39554814
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11564186/
Abstract

is a specialized intracellular parasitic pathogen capable of causing pneumonia, sinusitis, bronchitis, and other respiratory diseases, which pose significant public health challenges. Therefore, rapid, accurate, and sensitive diagnosis is crucial for the prevention and treatment of respiratory diseases caused by . In this study, we combined enzymatic recombination amplification (ERA) with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a system (CRISPR/Cas12a) to develop a dual detection platform termed the Cpn-ERA-CRISPR/Cas12a dual system. This system integrates both the ERA-CRISPR/Cas12a fluorescence system and the ERA-CRISPR/Cas12a lateral flow system. Detection results can be measured using a fluorescence detector or observed with the naked eye on lateral flow strips. The fluorescence system and the lateral flow system detect in 30 minutes and 15 minutes, respectively. This dual system exhibits no cross-reactivity with the other seven pathogens, demonstrating high specificity, and achieves a sensitivity of 10 copies/µL. Additionally, the Cpn-ERA-CRISPR/Cas12a dual system was employed to analyze 39 clinical samples, comprising 19 positive and 20 negative samples. The detection rate for positive samples was 100%, with no positive results in the negative samples, indicating a high level of concordance with qPCR results. In summary, the Cpn-ERA-CRISPR/Cas12a dual system represents a novel tool for diagnosing and holds promising application potential in grassroots community hospitals.

摘要

是一种专门的细胞内寄生病原体,能够引起肺炎、鼻窦炎、支气管炎和其他呼吸道疾病,对公共卫生构成重大挑战。因此,快速、准确、敏感的诊断对于预防和治疗 引起的呼吸道疾病至关重要。在本研究中,我们将酶促重组扩增(ERA)与成簇规律间隔短回文重复(CRISPR)/CRISPR 相关蛋白(Cas)12a 系统(CRISPR/Cas12a)相结合,开发了一种称为 Cpn-ERA-CRISPR/Cas12a 双检测平台。该系统集成了 ERA-CRISPR/Cas12a 荧光系统和 ERA-CRISPR/Cas12a 侧流系统。可以使用荧光检测器测量检测结果,也可以在侧流条上用肉眼观察。荧光系统和侧流系统分别在 30 分钟和 15 分钟内检测到 。该双系统与其他七种病原体无交叉反应,表现出高度特异性,灵敏度达到 10 拷贝/μL。此外,Cpn-ERA-CRISPR/Cas12a 双系统用于分析 39 个临床样本,包括 19 个阳性样本和 20 个阴性样本。阳性样本的检测率为 100%,阴性样本无阳性结果,与 qPCR 结果高度一致。综上所述,Cpn-ERA-CRISPR/Cas12a 双系统为 诊断提供了一种新工具,在基层社区医院具有广阔的应用潜力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/09892a46d5b2/fcimb-14-1477422-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/d8a3dfa93386/fcimb-14-1477422-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/b4f5448cad91/fcimb-14-1477422-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/c756ddafe8bc/fcimb-14-1477422-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/ac624f787235/fcimb-14-1477422-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/61708caed565/fcimb-14-1477422-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/09892a46d5b2/fcimb-14-1477422-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/d8a3dfa93386/fcimb-14-1477422-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/b4f5448cad91/fcimb-14-1477422-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/c756ddafe8bc/fcimb-14-1477422-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/ac624f787235/fcimb-14-1477422-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/61708caed565/fcimb-14-1477422-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1b2f/11564186/09892a46d5b2/fcimb-14-1477422-g006.jpg

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本文引用的文献

[1]
CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels.

Nat Commun. 2024-6-12

[2]
A rapid and sensitive CRISPR-Cas12a for the detection of .

Microbiol Spectr. 2024-2-6

[3]
Rapid detection of in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction.

J Clin Microbiol. 2024-1-17

[4]
Advancements in the synergy of isothermal amplification and CRISPR-cas technologies for pathogen detection.

Front Bioeng Biotechnol. 2023-10-10

[5]
CRISPR-Cas12a test strip (CRISPR/CAST) package: In-situ detection of Brucella from infected livestock.

BMC Vet Res. 2023-10-13

[6]
One-pot RPA-Cas12a assay for instant and visual detection of Burkholderia pseudomallei.

Anal Chim Acta. 2023-4-29

[7]
Sensitive Detection of by a Colorimetric Biosensor Based on Magnetic Separation and Rolling Circle Amplification.

Foods. 2022-6-23

[8]
Rapid and Visual Detection of Porcine Parvovirus Using an ERA-CRISPR/Cas12a System Combined With Lateral Flow Dipstick Assay.

Front Cell Infect Microbiol. 2022

[9]
Ultrasensitive, Specific, and Rapid Detection of Using the ERA/CRISPR-Cas12a Dual System.

Front Microbiol. 2022-5-13

[10]
Development of a Rapid and Efficient RPA-CRISPR/Cas12a Assay for Detection.

Front Microbiol. 2022-3-15

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