Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Sun, Chunrong, Jia, Nan, Huang, Xiaolan, Xiao, Fei, Zhou, Juan, Zhang, Yu, Fu, Jin, Xu, Zheng, Qu, Dong, Cui, Xiaodai, Wang, Yi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
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
_version_ 1784806063940304896
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
work_keys_str_mv AT sunchunrong realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT jianan realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT huangxiaolan realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT xiaofei realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT zhoujuan realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT zhangyu realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT fujin realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT xuzheng realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT qudong realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT cuixiaodai realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae
AT wangyi realtimemultiplecrossdisplacementamplificationassayforrapidandsensitivedetectionofhaemophilusinfluenzae