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Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System

Mycoplasma pneumoniae can cause severe respiratory tract infections and extrapulmonary diseases, which pose a significant threat to the health of children. Diagnostic methods for M. pneumoniae include isolation and culture, antibody detection, fluorescence quantitative PCR, and so on, but there are...

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Autores principales: Deng, Zhongliang, Hu, Haiyang, Tang, Dan, Liang, Jiaxin, Su, Xiaoling, Jiang, Tingqing, Hu, Xipan, Ying, Wanqin, Zhen, Deshuai, Xiao, Xilin, He, Jun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9136402/
https://www.ncbi.nlm.nih.gov/pubmed/35633705
http://dx.doi.org/10.3389/fmicb.2022.811768
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author Deng, Zhongliang
Hu, Haiyang
Tang, Dan
Liang, Jiaxin
Su, Xiaoling
Jiang, Tingqing
Hu, Xipan
Ying, Wanqin
Zhen, Deshuai
Xiao, Xilin
He, Jun
author_facet Deng, Zhongliang
Hu, Haiyang
Tang, Dan
Liang, Jiaxin
Su, Xiaoling
Jiang, Tingqing
Hu, Xipan
Ying, Wanqin
Zhen, Deshuai
Xiao, Xilin
He, Jun
author_sort Deng, Zhongliang
collection PubMed
description Mycoplasma pneumoniae can cause severe respiratory tract infections and extrapulmonary diseases, which pose a significant threat to the health of children. Diagnostic methods for M. pneumoniae include isolation and culture, antibody detection, fluorescence quantitative PCR, and so on, but there are various shortcomings in time, cost, convenience, and sensitivity. In this study, we developed a rapid, sensitive, specific, and economical method for the detection of M. pneumoniae, termed the ERA/CRISPR–Cas12a dual system. The system used the high specificity and collateral cleavage activity of the LbCas12a protein, combined with enzymatic recombination amplification (ERA) technology with strong amplification ability, allowing the results to be observed by a portable fluorometer or visualized by the naked eye with a dipstick, which could be obtained in approximately 30 min. The ERA/CRISPR–Cas12a fluorescence and dipstick system were able to detect M. pneumoniae at titers as low as 1 and 100 copies/μL, respectively. The specificity of the two interpretation methods was 100%, and no cross-reaction with other pathogens was observed. In the evaluation of 92 clinical samples, the positive predictive agreements of the ERA/CRISPR–Cas12a fluorescence and dipstick systems with qPCR detection were 100% and 92.86%, respectively. The negative predictive agreements of both methods were 100%. In conclusion, this study established a portable, rapid, low-cost, ultrasensitive, and specific method for the early and rapid diagnosis of M. pneumoniae to meet the needs of on-site rapid detection in primary health institutions.
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spelling pubmed-91364022022-05-28 Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System Deng, Zhongliang Hu, Haiyang Tang, Dan Liang, Jiaxin Su, Xiaoling Jiang, Tingqing Hu, Xipan Ying, Wanqin Zhen, Deshuai Xiao, Xilin He, Jun Front Microbiol Microbiology Mycoplasma pneumoniae can cause severe respiratory tract infections and extrapulmonary diseases, which pose a significant threat to the health of children. Diagnostic methods for M. pneumoniae include isolation and culture, antibody detection, fluorescence quantitative PCR, and so on, but there are various shortcomings in time, cost, convenience, and sensitivity. In this study, we developed a rapid, sensitive, specific, and economical method for the detection of M. pneumoniae, termed the ERA/CRISPR–Cas12a dual system. The system used the high specificity and collateral cleavage activity of the LbCas12a protein, combined with enzymatic recombination amplification (ERA) technology with strong amplification ability, allowing the results to be observed by a portable fluorometer or visualized by the naked eye with a dipstick, which could be obtained in approximately 30 min. The ERA/CRISPR–Cas12a fluorescence and dipstick system were able to detect M. pneumoniae at titers as low as 1 and 100 copies/μL, respectively. The specificity of the two interpretation methods was 100%, and no cross-reaction with other pathogens was observed. In the evaluation of 92 clinical samples, the positive predictive agreements of the ERA/CRISPR–Cas12a fluorescence and dipstick systems with qPCR detection were 100% and 92.86%, respectively. The negative predictive agreements of both methods were 100%. In conclusion, this study established a portable, rapid, low-cost, ultrasensitive, and specific method for the early and rapid diagnosis of M. pneumoniae to meet the needs of on-site rapid detection in primary health institutions. Frontiers Media S.A. 2022-05-13 /pmc/articles/PMC9136402/ /pubmed/35633705 http://dx.doi.org/10.3389/fmicb.2022.811768 Text en Copyright © 2022 Deng, Hu, Tang, Liang, Su, Jiang, Hu, Ying, Zhen, Xiao and He. 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 Microbiology
Deng, Zhongliang
Hu, Haiyang
Tang, Dan
Liang, Jiaxin
Su, Xiaoling
Jiang, Tingqing
Hu, Xipan
Ying, Wanqin
Zhen, Deshuai
Xiao, Xilin
He, Jun
Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System
title Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System
title_full Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System
title_fullStr Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System
title_full_unstemmed Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System
title_short Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System
title_sort ultrasensitive, specific, and rapid detection of mycoplasma pneumoniae using the era/crispr–cas12a dual system
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9136402/
https://www.ncbi.nlm.nih.gov/pubmed/35633705
http://dx.doi.org/10.3389/fmicb.2022.811768
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