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Padlock Probe-Based Nucleic Acid Amplification Tests: Point-of-care Diagnostics of Infectious Diseases
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Mats Nilsson)
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 [en]
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: urn:nbn:se:su:diva-167374ISBN: 978-91-7797-576-2 (print)ISBN: 978-91-7797-577-9 (electronic)OAI: oai:DiVA.org:su-167374DiVA, id: diva2:1299600
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
List of papers
1. Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection
Open this publication in new window or tab >>Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection
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2017 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 91, no 11, article id e00166-17Article in journal (Refereed) Published
Abstract [en]

An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.

Keywords
adenovirus, persistent infection, lytic infection, rolling-circle amplification, single-cell analysis
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-144695 (URN)10.1128/JVI.00166-17 (DOI)000402166500007 ()
Available from: 2017-07-21 Created: 2017-07-21 Last updated: 2019-03-29Bibliographically approved
2. A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses
Open this publication in new window or tab >>A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses
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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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-167318 (URN)10.1038/s41598-019-39854-3 (DOI)000459799800021 ()30814634 (PubMedID)
Available from: 2019-03-25 Created: 2019-03-25 Last updated: 2019-03-29Bibliographically approved
3. Multiplexed rolling circle amplification detection of Ebola, Zika and Dengue towards point-of-care diagnostics
Open this publication in new window or tab >>Multiplexed rolling circle amplification detection of Ebola, Zika and Dengue towards point-of-care diagnostics
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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
Molecular diagnostics, Infectious diseases, Tropical virus, Ebola virus disease, RCA, Membrane enrichment
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-167375 (URN)
Available from: 2019-03-27 Created: 2019-03-27 Last updated: 2019-03-28Bibliographically approved
4. The sweet detection of rolling circle amplification: Glucose-based electrochemical detection of virus nucleic acid
Open this publication in new window or tab >>The sweet detection of rolling circle amplification: Glucose-based electrochemical detection of virus nucleic acid
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Infectious diseases remain a constant threat on a global scale by recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralizing healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. Nucleic acid amplification technology (NAAT) has proven to be a powerful tool for the control of outbreak with high sensitivity and specificity. However, NAAT is mostly based on amplification methods that require specialized instrumentation and trained personnel, such as PCR with sophisticated detectors. Here, we present an isothermal padlock probe-based assay for the detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical approach as the read-out. The assay design is based on rolling circle amplification (RCA) upon magnetic beads, connecting the RCA products (RCPs) via streptavidin-biotin bridges to GOx needed for the electrochemical measurement with externally provided glucose. The RCPs forming on the surface of beads are imaged using scanning electron microscopy, and the presence of the GOx to the RCP complex is confirmed using atomic force microscopy. Parameters such as the choice of buffers, concentrations of glucose and GOx and measurement time were optimized, as well as the mode of addition of glucose was tested. 125 μg/mL of GOx with 5 mM glucose using PBS as washing buffer, monitored for 15 min were chosen as the optimized conditions. The effect of temperature was tested and found to be critical at 37 °C for enhanced performance of the sensor. Finally, we evaluate the analytical performance of our sensor system by using cell culture isolate and clinical samples of Ebola virus. The study provides a proof-of-concept of simple and portable molecular diagnostics for emerging pathogens, beneficial especially for resource-limited settings. 

Keywords
Molecular diagnostics, Infectious diseases, Ebola virus disease, RCA, Glucose Oxidase, Glucose sensing
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-167377 (URN)
Available from: 2019-03-27 Created: 2019-03-27 Last updated: 2019-03-28Bibliographically approved

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