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Multiplexed rolling circle amplification detection of Ebola, Zika and Dengue towards point-of-care diagnostics
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).
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent 2018-19 outbreak in Congo. Immediate diagnostic attention is demanded, and most importantly at the point-of-care in resource-limited settings. The performance and the operational parameters of conventional EBOV testing are limited by either their sensitivity, specificity, or both, and often do not cover other tropical disease viruses. We present a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV from cell culture isolates as well as clinical samples obtained from patients of West Africa outbreak. For this, a set of PLPs, separately targeting the vRNA and cRNA of all the seven genes of EBOV, were used in the RCA and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous duplex detection of both EBOV vRNA and cRNA was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. As PLPs and RCA are inherently mulitplexable, we demonstrate the extension of the probe panel to the simultaneous detection of the tropical viruses Ebola, Zika and Dengue. The simple, rapid, specific and multiplexable isothermal assay developed for tropical virus detection suits the point-of-care needs, bringing RCA a step closer to bedside diagnostics.

Keywords [en]
Molecular diagnostics, Infectious diseases, Tropical virus, Ebola virus disease, RCA, Membrane enrichment
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-167375OAI: oai:DiVA.org:su-167375DiVA, id: diva2:1299591
Available from: 2019-03-27 Created: 2019-03-27 Last updated: 2019-03-28Bibliographically approved
In thesis
1. Padlock Probe-Based Nucleic Acid Amplification Tests: Point-of-care Diagnostics of Infectious Diseases
Open this publication in new window or tab >>Padlock Probe-Based Nucleic Acid Amplification Tests: Point-of-care Diagnostics of Infectious Diseases
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Recent advancements in molecular biology and biotechnology have pushed the field of molecular diagnostics much further to benefit the society towards smart access for rapid and simplified health care. In this context, point-of-care (PoC) technologies that bring the inventions in diagnostics closer to bedside settings draw attention. This becomes all the more relevant in the case of infectious diseases which pose the major burden, in terms of mortality and economic loss, especially for third world developing countries with resource-limited settings (RLS). Moreover, emerging and re-emerging viruses, known for their rapid mutation rates, demand huge attention in terms of timely diagnosis and the need for effective treatments. Hence, appropriate and accurate tests to detect the pathogens with enhanced sensitivity and specificity would be needed to bridge the gap between bioanalytics and clinics.

This research work is an attempt to combine the tools and techniques required for the development of such efficient PoC technologies to combat infectious diseases. Among available nucleic acid-based amplification tests, padlock probing and isothermal rolling circle amplification are used to benefit from the advantages they offer for diagnostic applications, in terms of specificity, multiplexability, single molecule detection, high throughput, compatibility with various read-out platforms and inexpensive digital quantification.

In the first paper, simultaneous detection of RNA and DNA forms of adenovirus is shown to study the spatio-temporal expression patterns in both lytic and persistent infections. In situ quantification of viral DNA as well as transcripts with single cell resolution has been achieved. In the second paper, novel probe design strategy has been presented for the development of molecular assays to detect hypervariable RNA viruses. This approach becomes helpful in targeting rapidly evolving viruses by using mutation-tolerant probes for RCA. Third paper demonstrates simple RCA for rapid detection of Ebola virus in clinical samples, followed by a multiplexed detection with other re-emerging tropical viruses, namely Zika and Dengue. This study also includes a simple easy-to-operate pump-free membrane enrichment read-out, combined together with microscopy for digital quantification of the products. In the fourth paper, near point-of-care glucose sensor-based RCP detection has been proposed for Ebola virus detection. All these attempts clearly bring RCA closer to PoC settings for molecular diagnostics of virus infections.

Place, publisher, year, edition, pages
Department of Biochemistry and Biophysics, Stockholm University, 2019
Keywords
Nucleic Acid Amplification, Isothermal Amplification Methods, Padlock Probes, Rolling Circle Amplification, Molecular Diagnostics, Infectious Disease Diagnostics, Virus, Point-of-Care
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-167374 (URN)978-91-7797-576-2 (ISBN)978-91-7797-577-9 (ISBN)
Public defence
2019-05-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
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Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2019-04-17 Created: 2019-03-27 Last updated: 2019-04-09Bibliographically approved

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