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A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA 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
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 2872Article in journal (Refereed) Published
Abstract [en]

The establishment of a robust detection platform for RNA viruses still remains a challenge in molecular diagnostics due to their high mutation rates. Newcastle disease virus (NDV) is one such RNA avian virus with a hypervariable genome and multiple genotypes. Classical approaches like virus isolation, serology, immunoassays and RT-PCR are cumbersome, and limited in terms of specificity and sensitivity. Padlock probes (PLPs) are known for allowing the detection of multiple nucleic acid targets with high specificity, and in combination with Rolling circle amplification (RCA) have permitted the development of versatile pathogen detection assays. In this work, we aimed to detect hypervariable viruses by developing a novel PLP design strategy capable of tolerating mutations while preserving high specificity by targeting several moderately conserved regions and using degenerate bases. For this, we designed nine padlock probes based on the alignment of 335 sequences covering both Class I and II NDV. Our PLP design showed high coverage and specificity for the detection of eight out of ten reported genotypes of Class II NDV field isolated strains, yielding a detection limit of less than ten copies of viral RNA. Further taking advantage of the multiplex capability of PLPs, we successfully extended the assay for the simultaneous detection of three poultry RNA viruses (NDV, IBV and AIV) and combined it with a paper based microfluidic enrichment read-out for digital quantification. In summary, our novel PLP design addresses the current issue of tolerating mutations of highly emerging virus strains with high sensitivity and specificity.

Place, publisher, year, edition, pages
2019. Vol. 9, article id 2872
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-167318DOI: 10.1038/s41598-019-39854-3ISI: 000459799800021PubMedID: 30814634OAI: oai:DiVA.org:su-167318DiVA, id: diva2:1298960
Available from: 2019-03-25 Created: 2019-03-25 Last updated: 2019-03-29Bibliographically 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)
Opponent
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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