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Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii

Spotted fever group (SFG) rickettsioses are important zoonoses, threatening human health seriously and gradually attracting more attention in the world. SFG rickettsiae are classified as neglected pathogens. If these pathogens are detected at all, they are usually recognized very late in the infecti...

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Autores principales: Gui, Zheng, Cai, Hao, Wu, Lin, Miao, Qing, Yu, Jing feng, Cai, Ting, Mao, Rui
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9491570/
https://www.ncbi.nlm.nih.gov/pubmed/36084136
http://dx.doi.org/10.1371/journal.pntd.0010747
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author Gui, Zheng
Cai, Hao
Wu, Lin
Miao, Qing
Yu, Jing feng
Cai, Ting
Mao, Rui
author_facet Gui, Zheng
Cai, Hao
Wu, Lin
Miao, Qing
Yu, Jing feng
Cai, Ting
Mao, Rui
author_sort Gui, Zheng
collection PubMed
description Spotted fever group (SFG) rickettsioses are important zoonoses, threatening human health seriously and gradually attracting more attention in the world. SFG rickettsiae are classified as neglected pathogens. If these pathogens are detected at all, they are usually recognized very late in the infection through indirect detection of specific antibodies. Previous studies have shown that Rickettsia raoultii (R. raoultii), a member of the SFG rickettsiae, occurs with increasing incidence in remote countries. Therefore, a rapid detection method for R. raoultii is in urgently need. In this study, a R. raoultii diagnosis method by closed dumbbell-mediated isothermal amplification (R-CDA) assay targeting a conserved sequence of the outer membrane protein A (OmpA) gene with high sensitivity and specificity was developed. This assay offered a rapid and simple method for on-site detection of R. raoultii. Firstly, four pairs of R-CDA primers were designed and the optimum primer set was selected to amplify target gene specifically and effectively. Then, a pair of outer primer was designed to accelerate the reaction based on the inner primers to establish the RO-CDA reaction. In addition, the results of real-time amplification curves, melting curves and end-point colorimetric judgements showed that the established visual RO-CDA reaction could accurately detect R. raoultii without cross-reaction with other closely related pathogens. Furthermore, the detection limit of visual RO-CDA assay was 10 copies/μL, which was feasible for on-site detection with merits of easy-operation, rapidity, high sensitivity, and specificity. In conclusion, the developed RO-CDA detection method could be helpful for pathogen screening and epidemic prevention at the point of care.
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spelling pubmed-94915702022-09-22 Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii Gui, Zheng Cai, Hao Wu, Lin Miao, Qing Yu, Jing feng Cai, Ting Mao, Rui PLoS Negl Trop Dis Research Article Spotted fever group (SFG) rickettsioses are important zoonoses, threatening human health seriously and gradually attracting more attention in the world. SFG rickettsiae are classified as neglected pathogens. If these pathogens are detected at all, they are usually recognized very late in the infection through indirect detection of specific antibodies. Previous studies have shown that Rickettsia raoultii (R. raoultii), a member of the SFG rickettsiae, occurs with increasing incidence in remote countries. Therefore, a rapid detection method for R. raoultii is in urgently need. In this study, a R. raoultii diagnosis method by closed dumbbell-mediated isothermal amplification (R-CDA) assay targeting a conserved sequence of the outer membrane protein A (OmpA) gene with high sensitivity and specificity was developed. This assay offered a rapid and simple method for on-site detection of R. raoultii. Firstly, four pairs of R-CDA primers were designed and the optimum primer set was selected to amplify target gene specifically and effectively. Then, a pair of outer primer was designed to accelerate the reaction based on the inner primers to establish the RO-CDA reaction. In addition, the results of real-time amplification curves, melting curves and end-point colorimetric judgements showed that the established visual RO-CDA reaction could accurately detect R. raoultii without cross-reaction with other closely related pathogens. Furthermore, the detection limit of visual RO-CDA assay was 10 copies/μL, which was feasible for on-site detection with merits of easy-operation, rapidity, high sensitivity, and specificity. In conclusion, the developed RO-CDA detection method could be helpful for pathogen screening and epidemic prevention at the point of care. Public Library of Science 2022-09-09 /pmc/articles/PMC9491570/ /pubmed/36084136 http://dx.doi.org/10.1371/journal.pntd.0010747 Text en © 2022 Gui et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Gui, Zheng
Cai, Hao
Wu, Lin
Miao, Qing
Yu, Jing feng
Cai, Ting
Mao, Rui
Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii
title Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii
title_full Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii
title_fullStr Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii
title_full_unstemmed Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii
title_short Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii
title_sort visual closed dumbbell-mediated isothermal amplification (cda) for on-site detection of rickettsia raoultii
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9491570/
https://www.ncbi.nlm.nih.gov/pubmed/36084136
http://dx.doi.org/10.1371/journal.pntd.0010747
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