| 1. |
- Neumann, Felix, et al.
(författare)
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Isotachophoretically-driven rolling circle amplification unit for nucleic acid detection
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Ciftci, Sibel, 1987-, et al.
(författare)
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Multiplexed rolling circle amplification detection of Ebola, Zika and Dengue towards point-of-care diagnostics
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Ciftci, Sibel, 1987-, et al.
(författare)
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The sweet detection of rolling circle amplification : Glucose-based electrochemical detection of virus nucleic acid
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
- Hernández-Neuta, Iván, et al.
(författare)
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Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA processing in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification (RCA). This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions and high throughput capabilities, with flow rates up to 5 L/min without compromising performance. The obtained efficiency values using the fluidized bed were superior to a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based micro arrayand tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostic systems.
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| 5. |
- Lundin, Elin, 1983-, et al.
(författare)
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Factors affecting padlock probe efficiency
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Padlock probes have proved to be extremely versatile and useful molecular tools. They have unique properties that allow them to be used in various applications, ranging from diagnostic assays to spatially resolved transcriptomics. Padlock probes are used for detection of specific DNA or RNA sequences in enzymatic multistep assays. As the assays involve circularization and rolling circle amplification of the padlock probe, different factors play a role in the efficiency of the separate steps. Guidelines for how to design padlock probes have been lacking. We investigated how the length and the secondary structure of the different parts of the padlock probe affected its efficacy in the different steps of the assay as well as the impact on the total assay. The optimal length of the padlock probe is a compromise between a shorter total probe length, which leads to more efficient amplification and longer target specific sequence, which confers more efficient circularization. Complex secondary structure interfering with the detection motif or involving both the target-specific parts of the padlock probe seriously impair the assay efficiency. However, less complex secondary structures can be tolerated without significant efficiency loss. Taken together, the results present important considerations for the design of padlock probes and guidelines for how to improve the general detection efficiency.
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| 6. |
- Lundin, Elin, 1983-, et al.
(författare)
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Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. However, the spatiotemporal and diversification properties of RNA editing in the brain are largely unknown. Here, we applied in situ sequencing to distinguish between edited and unedited transcripts in distinct regions of the mouse brain at four developmental stages, and investigate the diversity of the RNA landscape.Results: We analyzed RNA editing at codon-altering sites using in situ sequencing at single-cell resolution, in combination with the detection of individual ADAR enzymes and specific cell type marker transcripts. This approach revealed cell-type specific regulation of RNA editing of a set of transcripts, and developmental and regional variation in editing levels for many of the targeted sites. We found increasing editing diversity throughout development, which arises through regional- and cell type-specific regulation of ADAR enzymes and target transcripts.Conclusions: Our single-cell in situ sequencing method has proved useful to study the complex landscape of RNA editing and our results indicate that this complexity arises due to distinct mechanisms of regulating individual RNA editing sites, acting both regionally and in specific cell types.
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| 7. |
- Qian, Xiaoyan, et al.
(författare)
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Target sequence design of padlock probes based on experimentally determined in situ synthesized cDNA fragments
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Padlock probes are widely used to target a short fragment of DNA. For example, in in situ sequencing (ISS), an image-based technology for highly multiplexed spatial gene expression analysis, cDNA target detection is mediated by padlock probes. Transcript counts from ISS generally has good correlation with next-generation sequencing read counts, but bias between different genes are also observed. Therefore, we developed a new method to isolate and sequence in situ synthesized cDNA and sought to use the read coverage information from it to guide padlock probe design. The results show limited correlation between cDNA library sequencing and ISS counts, but it can still help the probe design process by eliminating target sequences that are very unlikely to be detected. In addition, the method provides a way to systematically characterize in situ reverse transcription.
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| 8. |
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| 9. |
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| 10. |
- Horta, Sara, et al.
(författare)
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Evaluation of immuno-rolling circle amplification for multiplexed detection and profiling of antigen-specific antibody isotypes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Antibody characterization has become essential for diagnosis and development of therapeutic solutions in autoimmune, cardiovascular and infectious diseases. The specificity, affinity and isotype are crucial information for antibody studies and can be obtained from screening plasma samples or populations of B cells. Current technologies are mainly focusing on the discovery of abundant immunoglobulins, namely IgG, and are based on bulk measurements. In this study, we present a digital screening platform utilizing rolling circle amplification (RCA) for the detection of antigen-specific antibody isotypes in solution or secreted from single cells. To validate this approach, the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) was used as model disease and anti-ADAMTS13 antibodies were the target molecules. Antibody-oligonucleotide conjugates (AOCs) were designed for the multiplexed detection of human antibody isotypes IgA, IgG and IgM. Then, ADAMTS13 fragments were coated on glass slides and subsequently, target antibodies identified by specific AOC binding and visualized via an RCA assay. First, we validated the method by characterizing the assay specificity and limit of detection (LoD). When seeding different isotypes, we confirmed the high specificity of the assay (> 90%) and detection of monoclonal anti-ADAMTS13 IgG down to 0.3 ng/mL. A dilution series of a plasma sample of iTTP patient confirmed the multiplexed detection of the three isotypes with higher sensitivity compared to ELISA. Finally, we performed single cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving, achieving a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.
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