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A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application
Mycoplasma pneumoniae (MP), which is responsible for a majority of community-acquired pneumonia (CAP) in children, has been largely underestimated. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platf...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9887289/ https://www.ncbi.nlm.nih.gov/pubmed/36733967 http://dx.doi.org/10.3389/fbioe.2023.1022066 |
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author | Jia, Nan Zhou, Juan Xiao, Fei Zheng, Baoying Huang, Xiaolan Sun, Chunrong Fu, Jin Xu, Zheng Chen, Min Wang, Yi |
author_facet | Jia, Nan Zhou, Juan Xiao, Fei Zheng, Baoying Huang, Xiaolan Sun, Chunrong Fu, Jin Xu, Zheng Chen, Min Wang, Yi |
author_sort | Jia, Nan |
collection | PubMed |
description | Mycoplasma pneumoniae (MP), which is responsible for a majority of community-acquired pneumonia (CAP) in children, has been largely underestimated. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform termed MP-MCDA-CRISPR assay for MP infection diagnosis and clinical application. The MP-MCDA-CRISPR assay amplified the CARDS gene of MP by MCDA method, followed by trans-cleavage of the reporter molecular upon the formation of CRISPR-Cas12a-gRNA-target DNA complex, which was confirmed by the release of fluorescent signals. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the CARDS gene of MP. The optimal temperature for MCDA pre-amplification is 64°C, and the time for CRISPR-Cas12a-gRNA biosensing process is 5 min. The limit of detection (LoD) of the MP-MCDA-CRISPR assay is 50 fg per reaction without any cross-reaction with other non-MP pathogens. The MP-MCDA-CRISPR assay accurately identified the 50 real time-PCR positive clinical samples and 78 negative ones. Taken together, the MP-MCDA-CRISPR assay designed here is a promising diagnostic tool for point-of care (POC) testing of MP infection. |
format | Online Article Text |
id | pubmed-9887289 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-98872892023-02-01 A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application Jia, Nan Zhou, Juan Xiao, Fei Zheng, Baoying Huang, Xiaolan Sun, Chunrong Fu, Jin Xu, Zheng Chen, Min Wang, Yi Front Bioeng Biotechnol Bioengineering and Biotechnology Mycoplasma pneumoniae (MP), which is responsible for a majority of community-acquired pneumonia (CAP) in children, has been largely underestimated. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform termed MP-MCDA-CRISPR assay for MP infection diagnosis and clinical application. The MP-MCDA-CRISPR assay amplified the CARDS gene of MP by MCDA method, followed by trans-cleavage of the reporter molecular upon the formation of CRISPR-Cas12a-gRNA-target DNA complex, which was confirmed by the release of fluorescent signals. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the CARDS gene of MP. The optimal temperature for MCDA pre-amplification is 64°C, and the time for CRISPR-Cas12a-gRNA biosensing process is 5 min. The limit of detection (LoD) of the MP-MCDA-CRISPR assay is 50 fg per reaction without any cross-reaction with other non-MP pathogens. The MP-MCDA-CRISPR assay accurately identified the 50 real time-PCR positive clinical samples and 78 negative ones. Taken together, the MP-MCDA-CRISPR assay designed here is a promising diagnostic tool for point-of care (POC) testing of MP infection. Frontiers Media S.A. 2023-01-17 /pmc/articles/PMC9887289/ /pubmed/36733967 http://dx.doi.org/10.3389/fbioe.2023.1022066 Text en Copyright © 2023 Jia, Zhou, Xiao, Zheng, Huang, Sun, Fu, Xu, Chen and Wang. 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 | Bioengineering and Biotechnology Jia, Nan Zhou, Juan Xiao, Fei Zheng, Baoying Huang, Xiaolan Sun, Chunrong Fu, Jin Xu, Zheng Chen, Min Wang, Yi A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application |
title | A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application |
title_full | A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application |
title_fullStr | A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application |
title_full_unstemmed | A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application |
title_short | A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application |
title_sort | crispr-cas12a—based platform for ultrasensitive, rapid, and highly specific detection of mycoplasma pneumonia in clinical application |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9887289/ https://www.ncbi.nlm.nih.gov/pubmed/36733967 http://dx.doi.org/10.3389/fbioe.2023.1022066 |
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