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基于 RPA-CRISPR/Cas12a 的快速超灵敏双读取检测平台。

A rapid and ultra-sensitive dual readout platform for detection based on RPA-CRISPR/Cas12a.

机构信息

Center for Medical Laboratory Science, Affiliated Hospital of Youjiang Medical University for Nationalities, Guangxi, China.

Baise Key Laboratory for Research and Development on Clinical Molecular Diagnosis for High-Incidence Diseases, Guangxi, China.

出版信息

Front Cell Infect Microbiol. 2024 Jun 27;14:1362513. doi: 10.3389/fcimb.2024.1362513. eCollection 2024.


DOI:10.3389/fcimb.2024.1362513
PMID:38994004
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11236598/
Abstract

The bacterium () was the primary pathogen of hospital-acquired infection, but the current detection method could not rapidly and conveniently identify . Recombinase polymerase amplification (RPA) was a fast and convenient isothermal amplification technology, and the clustered regularly interspaced short palindromic repeats (CRISPR) system could rapidly amplify the signal of RPA and improve its limit of detection (LOD). In this study, we designed three pairs of RPA primers for the rcsA gene of , amplified the RPA signal through single-strand DNA reporter cleavage by CRISPR/Cas12a, and finally analyzed the cleavage signal using fluorescence detection (FD) and lateral flow test strips (LFTS). Our results indicated that the RPA-CRISPR/Cas12a platform could specifically identify from eleven common clinical pathogens. The LOD of FD and LFTS were 1 fg/μL and 10 fg/μL, respectively. In clinical sample testing, the RPA-CRISPR/Cas12a platform was consistent with the culture method and qPCR method, and its sensitivity and specificity were 100% (16/16) and 100% (9/9), respectively. With the advantages of detection speed, simplicity, and accuracy, the RPA-CRISPR/Cas12a platform was expected to be a convenient tool for the early clinical detection of .

摘要

该细菌()是医院获得性感染的主要病原体,但目前的检测方法无法快速、方便地识别。重组酶聚合酶扩增(RPA)是一种快速、方便的等温扩增技术,而簇状规则间隔短回文重复序列(CRISPR)系统可以快速扩增 RPA 的信号,提高其检测限(LOD)。在这项研究中,我们设计了三对针对的 rcsA 基因的 RPA 引物,通过 CRISPR/Cas12a 对单链 DNA 报告分子的切割来扩增 RPA 信号,最后通过荧光检测(FD)和侧向流测试条(LFTS)分析切割信号。我们的结果表明,RPA-CRISPR/Cas12a 平台可以特异性地从 11 种常见的临床病原体中识别出。FD 和 LFTS 的 LOD 分别为 1 fg/μL 和 10 fg/μL。在临床样本检测中,RPA-CRISPR/Cas12a 平台与培养法和 qPCR 法一致,其灵敏度和特异性分别为 100%(16/16)和 100%(9/9)。由于具有检测速度快、操作简单、准确性高等优点,RPA-CRISPR/Cas12a 平台有望成为一种方便的临床早期检测工具。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/2fe97fefcc22/fcimb-14-1362513-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/34cce4ba75b6/fcimb-14-1362513-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/1bde158062bc/fcimb-14-1362513-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/4ca1a44ec3c0/fcimb-14-1362513-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/4d28206faa38/fcimb-14-1362513-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/573bf0088c07/fcimb-14-1362513-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/6eb6a02f608d/fcimb-14-1362513-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/2fe97fefcc22/fcimb-14-1362513-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/34cce4ba75b6/fcimb-14-1362513-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/1bde158062bc/fcimb-14-1362513-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/4ca1a44ec3c0/fcimb-14-1362513-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/4d28206faa38/fcimb-14-1362513-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/573bf0088c07/fcimb-14-1362513-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/6eb6a02f608d/fcimb-14-1362513-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/613f/11236598/2fe97fefcc22/fcimb-14-1362513-g007.jpg

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引用本文的文献

[1]
Rapid detection of by glycerol one-pot RAA/CRISPR-Cas12a method.

Front Chem. 2025-7-25

[2]
Recombinase Polymerase Amplification (RPA)-ELISA as an Isothermal Molecular POCT Method for Bacterial Respiratory Infection Diagnosis.

Avicenna J Med Biotechnol. 2025

本文引用的文献

[1]
Multiple accurate and sensitive arrays for Capripoxvirus (CaPV) differentiation.

Anal Chim Acta. 2023-8-1

[2]
Highly Sensitive and Specific Detection of Mobilized Colistin Resistance Gene by CRISPR-Based Platform.

Microbiol Spectr. 2022-10-26

[3]
A CRISPR-Cas12a-based diagnostic method for multiple genotypes of severe fever with thrombocytopenia syndrome virus.

PLoS Negl Trop Dis. 2022-8

[4]
One-Pot Isothermal LAMP-CRISPR-Based Assay for Klebsiella pneumoniae Detection.

Microbiol Spectr. 2022-8-31

[5]
One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a.

Appl Microbiol Biotechnol. 2022-6

[6]
Sensitive and high-accuracy detection of Salmonella based on CRISPR/Cas12a combined with recombinase polymerase amplification.

Lett Appl Microbiol. 2022-10

[7]
Rapid Detection of Carrying Virulence Gene by Recombinase Polymerase Amplification Combined With Lateral Flow Strips.

Front Cell Infect Microbiol. 2022

[8]
Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay.

Anal Chem. 2022-6-14

[9]
Development of a RPA-CRISPR-Cas12a Assay for Rapid, Simple, and Sensitive Detection of .

Front Microbiol. 2022-4-8

[10]
Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant .

Diagnostics (Basel). 2022-3-28

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