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Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae
Haemophilus influenzae is an opportunistic pathogen usually causing bacteremia, meningitis, and pneumonia in children. Here, we developed a method based on multiple cross displacement amplification (MCDA) method and real-tme fluorescence technique for rapid detection of H. influenzae. A set of 10 pr...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9551287/ https://www.ncbi.nlm.nih.gov/pubmed/36237430 http://dx.doi.org/10.3389/fcimb.2022.1004183 |
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author | Sun, Chunrong Jia, Nan Huang, Xiaolan Xiao, Fei Zhou, Juan Zhang, Yu Fu, Jin Xu, Zheng Qu, Dong Cui, Xiaodai Wang, Yi |
author_facet | Sun, Chunrong Jia, Nan Huang, Xiaolan Xiao, Fei Zhou, Juan Zhang, Yu Fu, Jin Xu, Zheng Qu, Dong Cui, Xiaodai Wang, Yi |
author_sort | Sun, Chunrong |
collection | PubMed |
description | Haemophilus influenzae is an opportunistic pathogen usually causing bacteremia, meningitis, and pneumonia in children. Here, we developed a method based on multiple cross displacement amplification (MCDA) method and real-tme fluorescence technique for rapid detection of H. influenzae. A set of 10 primers was designed for the H. influenzae real-time MCDA reaction, and a core primer was modified with a restriction endonuclease recognition sequence, a fluorescent, and a quencher according to the principle of the real-time MCDA assay. The H. influenzae real-time MCDA reactions were performed using a fluorescence instrument at 63°C for 40 min. The H. influenzae real-time MCDA assay can specifically detect H. influenzae without any cross-reaction with other bacteria as our results confirmed. The sensitivity of our assay is as low as 10 CFU per reaction. To validate its feasibility, our assay was applied to 40 DNA extracted from sputum samples. The results obtained from H. influenzae real-time MCDA were compared with that of H. influenzae–loop-mediated isothermal amplification (H. influenzae-LAMP) and MCDA-based lateral flow biosensor (MCDA-LFB). The positive rate of the real-time MCDA assay was 62.5%, which was consistent with the H. influenzae-MCDA-LFB assay, but was more sensitive than H. influenzae-LAMP (57.5%). Furthermore, the H. influenzae real-time MCDA assay takes only 40 min, which was less than that of a traditional PCR test. Taken together, the H. influenzae real-time MCDA assay reported here offers a new and valuable diagnostic tool for the reliable and rapid detection of H. influenzae. |
format | Online Article Text |
id | pubmed-9551287 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-95512872022-10-12 Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae Sun, Chunrong Jia, Nan Huang, Xiaolan Xiao, Fei Zhou, Juan Zhang, Yu Fu, Jin Xu, Zheng Qu, Dong Cui, Xiaodai Wang, Yi Front Cell Infect Microbiol Cellular and Infection Microbiology Haemophilus influenzae is an opportunistic pathogen usually causing bacteremia, meningitis, and pneumonia in children. Here, we developed a method based on multiple cross displacement amplification (MCDA) method and real-tme fluorescence technique for rapid detection of H. influenzae. A set of 10 primers was designed for the H. influenzae real-time MCDA reaction, and a core primer was modified with a restriction endonuclease recognition sequence, a fluorescent, and a quencher according to the principle of the real-time MCDA assay. The H. influenzae real-time MCDA reactions were performed using a fluorescence instrument at 63°C for 40 min. The H. influenzae real-time MCDA assay can specifically detect H. influenzae without any cross-reaction with other bacteria as our results confirmed. The sensitivity of our assay is as low as 10 CFU per reaction. To validate its feasibility, our assay was applied to 40 DNA extracted from sputum samples. The results obtained from H. influenzae real-time MCDA were compared with that of H. influenzae–loop-mediated isothermal amplification (H. influenzae-LAMP) and MCDA-based lateral flow biosensor (MCDA-LFB). The positive rate of the real-time MCDA assay was 62.5%, which was consistent with the H. influenzae-MCDA-LFB assay, but was more sensitive than H. influenzae-LAMP (57.5%). Furthermore, the H. influenzae real-time MCDA assay takes only 40 min, which was less than that of a traditional PCR test. Taken together, the H. influenzae real-time MCDA assay reported here offers a new and valuable diagnostic tool for the reliable and rapid detection of H. influenzae. Frontiers Media S.A. 2022-09-27 /pmc/articles/PMC9551287/ /pubmed/36237430 http://dx.doi.org/10.3389/fcimb.2022.1004183 Text en Copyright © 2022 Sun, Jia, Huang, Xiao, Zhou, Zhang, Fu, Xu, Qu, Cui 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 | Cellular and Infection Microbiology Sun, Chunrong Jia, Nan Huang, Xiaolan Xiao, Fei Zhou, Juan Zhang, Yu Fu, Jin Xu, Zheng Qu, Dong Cui, Xiaodai Wang, Yi Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae |
title | Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae |
title_full | Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae |
title_fullStr | Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae |
title_full_unstemmed | Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae |
title_short | Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae |
title_sort | real-time multiple cross displacement amplification assay for rapid and sensitive detection of haemophilus influenzae |
topic | Cellular and Infection Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9551287/ https://www.ncbi.nlm.nih.gov/pubmed/36237430 http://dx.doi.org/10.3389/fcimb.2022.1004183 |
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