Back to Search Start Over

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

Authors :
Zheng Gui
Hao Cai
Lin Wu
Qing Miao
Jing Feng Yu
Ting Cai
Rui Mao
Source :
PLoS Neglected Tropical Diseases, Vol 16, Iss 9, p e0010747 (2022)
Publication Year :
2022
Publisher :
Public Library of Science (PLoS), 2022.

Abstract

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.

Details

Language :
English
ISSN :
19352727 and 19352735
Volume :
16
Issue :
9
Database :
Directory of Open Access Journals
Journal :
PLoS Neglected Tropical Diseases
Publication Type :
Academic Journal
Accession number :
edsdoj.36eb965a55ce4a489484cae761de7022
Document Type :
article
Full Text :
https://doi.org/10.1371/journal.pntd.0010747