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Digital Rolling Circle Amplification-Based Detection of Ebola and Other Tropical Viruses
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).ORCID iD: 0000-0003-3746-3693
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Number of Authors: 72020 (English)In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 22, no 2, p. 272-283Article in journal (Refereed) Published
Abstract [en]

Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent in 2018 to 2019 in Congo. Thus, immediate diagnostic attention is demanded at the point of care in resource-limited settings, because the performance and the operational parameters of conventional EBOV testing are Limited. Especially, their sensitivity, specificity, and coverage of other tropical disease viruses make them unsuitable for diagnostic at the point of care. Here, a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV is presented. For this, a set of PLPs, separately targeting the viral RNA and complementary RNA of all seven EBOV genes, was used in the RCA assay and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous detection of both EBOV RNA types was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. Because PLPs and RCA are inherently multiplexable, we demonstrate the extension of the probe panel for the simultaneous detection of the tropical viruses Ebola, Zika, and Dengue. The demonstrated high specificity, sensitivity, and multiplexing capability in combination with the digital quantification rendered the assay a promising diagnostic tool toward tropical virus detection at the point of care.

Place, publisher, year, edition, pages
2020. Vol. 22, no 2, p. 272-283
National Category
Biological Sciences Microbiology in the medical area
Identifiers
URN: urn:nbn:se:su:diva-180468DOI: 10.1016/j.jmoldx.2019.10.014ISI: 000514249000015PubMedID: 31837428OAI: oai:DiVA.org:su-180468DiVA, id: diva2:1422381
Available from: 2020-04-07 Created: 2020-04-07 Last updated: 2022-03-23Bibliographically approved
In thesis
1. Advancing isothermal nucleic acid amplification tests: Towards democratization of diagnostics
Open this publication in new window or tab >>Advancing isothermal nucleic acid amplification tests: Towards democratization of diagnostics
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Modern healthcare is the result of scientific advancement across disciplines and has enabled us to understand the rationale behind many diseases and how to treat or cure them; but still a myriad of unanswered questions remains. Especially infectious diseases play an important role in healthcare as they pose a constant threat for global health and well-being. This was painfully highlighted in this year's ongoing COVID-19 pandemic with more than 40 million people infected and over 1 million deaths. Pandemics like this have not only devastating effects on global health but also economy.

Therefore, scientific research in the field of infectious diseases is paramount to ensure outbreak control and surveillance of emerging threats. Current healthcare relies heavily on the diagnosis of infectious diseases in centralized healthcare centers thereby overlooking the access of molecular diagnostics for other areas such as airports, home-testing and especially the developing world with its limited resources. Towards this, various isothermal nucleic acid amplification technologies have been developed that hold the promise to bring state-of-the-art molecular diagnostics into these areas as they are versatile, sensitive and specific, and cost-effective. One such technique is rolling circle amplification which was used in this thesis.

This research work provides an overview of the developments in biochemistry, related disciplines and their combination to design methods for diagnostic platforms tackling infectious diseases. The studies conducted in this work can be considered as individual modules for addressing challenges, like typing of pathogens and disease-related antibodies, and inexpensive bulk as well as digital quantification and simplified assay schemes. These approaches and their combinations aim to bring rolling circle amplification-based assay schemes into the molecular diagnostic field and towards decentralized healthcare.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. p. 68
Keywords
molecular diagnostics, infectious diseases, point-of-care, digital quantification, fluorescence detection, rolling circle amplification, padlock probes, microfluidic enrichment
National Category
Biochemistry Molecular Biology Infectious Medicine Microbiology in the medical area
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-185978 (URN)978-91-7911-317-9 (ISBN)978-91-7911-318-6 (ISBN)
Public defence
2020-12-10, Pascal, Science for Life Laboratory, Tomtebodavägen 23A or digitally via Zoom: https://stockholmuniversity.zoom.us/j/64931329555, Solna, 16:00 (English)
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Supervisors
Available from: 2020-11-17 Created: 2020-10-27 Last updated: 2025-02-20Bibliographically approved

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Ciftci, SibelNeumann, FelixNilsson, MatsMadaboosi, Narayanan

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