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Isotachophoretically-driven rolling circle amplification unit for nucleic acid detection
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]

Nucleic acid amplification tests have revolutionized the biomedical field by offering high sensitivity and specificity. Polymerase chain reaction (PCR) is considered as the gold standard for nucleic acid amplification; however, it requires sophisticated instrumentation for temperature cycling and real-time detection which makes it expensive. Isothermal amplification technologies have been developed to overcome these drawbacks, such as rolling circle amplification (RCA). In this work, we use the RCA and combine it with isotachophoresis (ITP) to increase the sensitivity for fluorescent real-time detection of nucleic acid amplification. For this, we use a top-down approach by first developing a suitable buffer system that supports RCA and ITP, and subsequently show the focusing of differently-sized and concentrated RCA products. Next, we compare our ITP-RCA assay with a commercial instrument for real-time fluorescence monitoring and demonstrate higher sensitivity from our method. Finally, we aim to combine the ligation and amplification step into ITP to simplify the RCA assay into a one-step reaction. The presented combination of RCA with ITP opens up new opportunities by making nucleic acid detection faster and simpler with potential applications for molecular diagnostics of infectious diseases.

National Category
Biochemistry Molecular Biology
Research subject
Biochemistry; Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-185967OAI: oai:DiVA.org:su-185967DiVA, id: diva2:1477847
Available from: 2020-10-20 Created: 2020-10-20 Last updated: 2025-02-20Bibliographically 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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Neumann, FelixMadaboosi, NarayananNilsson, Mats

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