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Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system

Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen involved in a wide range of infections and antimicrobial resistance. Rapid and reliable diagnostic methods are of vital important for early identification, treatment, and stop of P. aeruginosa infections. In this study, we dev...

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Autores principales: Liu, Shuang, Huang, Siyuan, Li, Fang, Sun, Yuanyuan, Fu, Jin, Xiao, Fei, Jia, Nan, Huang, Xiaolan, Sun, Chunrong, Zhou, Juan, Wang, Yi, Qu, Dong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10449609/
https://www.ncbi.nlm.nih.gov/pubmed/37637458
http://dx.doi.org/10.3389/fcimb.2023.1239269
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author Liu, Shuang
Huang, Siyuan
Li, Fang
Sun, Yuanyuan
Fu, Jin
Xiao, Fei
Jia, Nan
Huang, Xiaolan
Sun, Chunrong
Zhou, Juan
Wang, Yi
Qu, Dong
author_facet Liu, Shuang
Huang, Siyuan
Li, Fang
Sun, Yuanyuan
Fu, Jin
Xiao, Fei
Jia, Nan
Huang, Xiaolan
Sun, Chunrong
Zhou, Juan
Wang, Yi
Qu, Dong
author_sort Liu, Shuang
collection PubMed
description Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen involved in a wide range of infections and antimicrobial resistance. Rapid and reliable diagnostic methods are of vital important for early identification, treatment, and stop of P. aeruginosa infections. In this study, we developed a simple, rapid, sensitive, and specific detection platform for P. aeruginosa infection diagnosis. The method integrated recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 12a (Cas12a) biosensing system and was termed P. aeruginosa–CRISPR–RPA assay. The P. aeruginosa–CRISPR–RPA assay was subject to optimization of reaction conditions and evaluation of sensitivity, specificity, and clinical feasibility with the serial dilutions of P. aeruginosa genomic DNA, the non–P. aeruginosa strains, and the clinical samples. As a result, the P. aeruginosa–CRISPR–RPA assay was able to complete P. aeruginosa detection within half an hour, including RPA reaction at 42°C for 20 min and CRISPR-Cas12a detection at 37°C for 10 min. The diagnostic method exhibited high sensitivity (60 fg per reaction, ~8 copies) and specificity (100%). The results of the clinical samples by P. aeruginosa–CRISPR–RPA assay were consistent to that of the initial result by microfluidic chip method. These data demonstrated that the newly developed P. aeruginosa–CRISPR–RPA assay was reliable for P. aeruginosa detection. In summary, the P. aeruginosa–CRISPR–RPA assay is a promising tool to early and rapid diagnose P. aeruginosa infection and stop its wide spread especially in the hospital settings.
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spelling pubmed-104496092023-08-26 Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system Liu, Shuang Huang, Siyuan Li, Fang Sun, Yuanyuan Fu, Jin Xiao, Fei Jia, Nan Huang, Xiaolan Sun, Chunrong Zhou, Juan Wang, Yi Qu, Dong Front Cell Infect Microbiol Cellular and Infection Microbiology Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen involved in a wide range of infections and antimicrobial resistance. Rapid and reliable diagnostic methods are of vital important for early identification, treatment, and stop of P. aeruginosa infections. In this study, we developed a simple, rapid, sensitive, and specific detection platform for P. aeruginosa infection diagnosis. The method integrated recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 12a (Cas12a) biosensing system and was termed P. aeruginosa–CRISPR–RPA assay. The P. aeruginosa–CRISPR–RPA assay was subject to optimization of reaction conditions and evaluation of sensitivity, specificity, and clinical feasibility with the serial dilutions of P. aeruginosa genomic DNA, the non–P. aeruginosa strains, and the clinical samples. As a result, the P. aeruginosa–CRISPR–RPA assay was able to complete P. aeruginosa detection within half an hour, including RPA reaction at 42°C for 20 min and CRISPR-Cas12a detection at 37°C for 10 min. The diagnostic method exhibited high sensitivity (60 fg per reaction, ~8 copies) and specificity (100%). The results of the clinical samples by P. aeruginosa–CRISPR–RPA assay were consistent to that of the initial result by microfluidic chip method. These data demonstrated that the newly developed P. aeruginosa–CRISPR–RPA assay was reliable for P. aeruginosa detection. In summary, the P. aeruginosa–CRISPR–RPA assay is a promising tool to early and rapid diagnose P. aeruginosa infection and stop its wide spread especially in the hospital settings. Frontiers Media S.A. 2023-08-10 /pmc/articles/PMC10449609/ /pubmed/37637458 http://dx.doi.org/10.3389/fcimb.2023.1239269 Text en Copyright © 2023 Liu, Huang, Li, Sun, Fu, Xiao, Jia, Huang, Sun, Zhou, Wang and Qu https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular and Infection Microbiology
Liu, Shuang
Huang, Siyuan
Li, Fang
Sun, Yuanyuan
Fu, Jin
Xiao, Fei
Jia, Nan
Huang, Xiaolan
Sun, Chunrong
Zhou, Juan
Wang, Yi
Qu, Dong
Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system
title Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system
title_full Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system
title_fullStr Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system
title_full_unstemmed Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system
title_short Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system
title_sort rapid detection of pseudomonas aeruginosa by recombinase polymerase amplification combined with crispr-cas12a biosensing system
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10449609/
https://www.ncbi.nlm.nih.gov/pubmed/37637458
http://dx.doi.org/10.3389/fcimb.2023.1239269
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