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...
Published in: | PLOS Neglected Tropical Diseases |
---|---|
Main Authors: | , , , , , , |
Format: | Article in Journal/Newspaper |
Language: | English |
Published: |
Public Library of Science (PLoS)
2022
|
Subjects: | |
Online Access: | https://doi.org/10.1371/journal.pntd.0010747 https://doaj.org/article/36eb965a55ce4a489484cae761de7022 |
id |
ftdoajarticles:oai:doaj.org/article:36eb965a55ce4a489484cae761de7022 |
---|---|
record_format |
openpolar |
spelling |
ftdoajarticles:oai:doaj.org/article:36eb965a55ce4a489484cae761de7022 2023-05-15T15:11:50+02:00 Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii. Zheng Gui Hao Cai Lin Wu Qing Miao Jing Feng Yu Ting Cai Rui Mao 2022-09-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0010747 https://doaj.org/article/36eb965a55ce4a489484cae761de7022 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0010747 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0010747 https://doaj.org/article/36eb965a55ce4a489484cae761de7022 PLoS Neglected Tropical Diseases, Vol 16, Iss 9, p e0010747 (2022) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2022 ftdoajarticles https://doi.org/10.1371/journal.pntd.0010747 2022-12-30T19:57:32Z 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. Article in Journal/Newspaper Arctic Human health Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 16 9 e0010747 |
institution |
Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Zheng Gui Hao Cai Lin Wu Qing Miao Jing Feng Yu Ting Cai Rui Mao Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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. |
format |
Article in Journal/Newspaper |
author |
Zheng Gui Hao Cai Lin Wu Qing Miao Jing Feng Yu Ting Cai Rui Mao |
author_facet |
Zheng Gui Hao Cai Lin Wu Qing Miao Jing Feng Yu Ting Cai Rui Mao |
author_sort |
Zheng Gui |
title |
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_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_sort |
visual closed dumbbell-mediated isothermal amplification (cda) for on-site detection of rickettsia raoultii. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2022 |
url |
https://doi.org/10.1371/journal.pntd.0010747 https://doaj.org/article/36eb965a55ce4a489484cae761de7022 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic Human health |
genre_facet |
Arctic Human health |
op_source |
PLoS Neglected Tropical Diseases, Vol 16, Iss 9, p e0010747 (2022) |
op_relation |
https://doi.org/10.1371/journal.pntd.0010747 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0010747 https://doaj.org/article/36eb965a55ce4a489484cae761de7022 |
op_doi |
https://doi.org/10.1371/journal.pntd.0010747 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
16 |
container_issue |
9 |
container_start_page |
e0010747 |
_version_ |
1766342616576688128 |