| 1. |
- Hernández-Neuta, Iván, 1986-, et al.
(författare)
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Highly multiplexed targeted sequencing strategy for infectious disease surveillance
- 2023
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Ingår i: BMC Biotechnology. - 1472-6750. ; 23
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Tidskriftsartikel (refereegranskat)abstract
- Background Global efforts to characterize diseases of poverty are hampered by lack of affordable and comprehensive detection platforms, resulting in suboptimal allocation of health care resources and inefficient disease control. Next generation sequencing (NGS) can provide accurate data and high throughput. However, shotgun and metagenome-based NGS approaches are limited by low concentrations of microbial DNA in clinical samples, requirements for tailored sample and library preparations plus extensive bioinformatics analysis. Here, we adapted molecular inversion probes (MIPs) as a cost-effective target enrichment approach to characterize microbial infections from blood samples using short-read sequencing. We designed a probe panel targeting 2 bacterial genera, 21 bacterial and 6 fungi species and 7 antimicrobial resistance markers (AMRs).Results Our approach proved to be highly specific to detect down to 1 in a 1000 pathogen DNA targets contained in host DNA. Additionally, we were able to accurately survey pathogens and AMRs in 20 out of 24 samples previously profiled with routine blood culture for sepsis.Conclusions Overall, our targeted assay identifies microbial pathogens and AMRs with high specificity at high throughput, without the need for extensive sample preparation or bioinformatics analysis, simplifying its application for characterization and surveillance of infectious diseases in medium- to low- resource settings.
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| 2. |
- Ciftci, Sibel, et al.
(författare)
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A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Dou, Dan, et al.
(författare)
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Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method
- 2017
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 20:1, s. 251-263
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Tidskriftsartikel (refereegranskat)abstract
- Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.
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| 4. |
- Guo, Maoxiang, et al.
(författare)
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Cross-membrane electrical detection of DNA
- 2017
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Ingår i: Proceedings 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017. - : The Chemical and Biological Microsystems Society (CBMS).
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Konferensbidrag (refereegranskat)abstract
- We introduce out-of-plane metallic nanowire formation on DNA templates, which are stretched through a porous membrane by applying a receding meniscus interface. We demonstrate the direct electrical detection of DNA using these gold nanowire bridges between the membrane’s opposite surfaces. Such a simple electrical readout can be extended for biosensor applications, thanks to the high specificity and multiplexing offered by Rolling Circle Amplification (RCA).
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| 5. |
- Guo, Maoxiang, et al.
(författare)
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Efficient DNA-assisted synthesis of trans-membrane gold nanowires
- 2018
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Ingår i: microsystems and nanoengineering. - : Springer Science and Business Media LLC. - 2055-7434. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Whereas electric circuits and surface-based (bio) chemical sensors are mostly constructed in-plane due to ease of manufacturing, 3D microscale and nanoscale structures allow denser integration of electronic components and improved mass transport of the analyte to (bio) chemical sensor surfaces. This work reports the first out-of-plane metallic nanowire formation based on stretching of DNA through a porous membrane. We use rolling circle amplification (RCA) to generate long single-stranded DNA concatemers with one end anchored to the surface. The DNA strands are stretched through the pores in the membrane during liquid removal by forced convection. Because the liquid-air interface movement across the membrane occurs in every pore, DNA stretching across the membrane is highly efficient. The stretched DNA molecules are transformed into trans-membrane gold nanowires through gold nanoparticle hybridization and gold enhancement chemistry. A 50 fM oligonucleotide concentration, a value two orders of magnitude lower than previously reported for flat surface-based nanowire formation, was sufficient for nanowire formation. We observed nanowires in up to 2.7% of the membrane pores, leading to an across-membrane electrical conductivity reduction from open circuit to <20 Omega. The simple electrical read-out offers a high signal-to-noise ratio and can also be extended for use as a biosensor due to the high specificity and scope for multiplexing offered by RCA.
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| 6. |
- 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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| 7. |
- Hernández-Neuta, Iván, et al.
(författare)
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Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode
- 2018
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 102, s. 531-539
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Tidskriftsartikel (refereegranskat)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 analysis 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. 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 with high throughput processing up to 120 mu L of DNA dilution at flow rates ranging from 1 to 5 mu L/min without compromising performance. The fluidized bed was 20-50% more efficient than 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 microarray and 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 diagnostics.
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| 8. |
- Hernández-Neuta, Iván, 1986-
(författare)
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Nucleic acid analysis tools : Novel technologies and biomedical applications
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nucleic acids are fundamental molecules of living organisms functioning essentially as the molecular information carriers of life. From how an organism is built to how it responds to external conditions, all of it, can be found in the form of nucleic acid sequences inside every single cell of every life form on earth.Therefore, accessing these sequences provides key information regarding the molecular identity and functional state of any living organism, this is very useful for areas like biomedicine, where accessing and understanding these molecular signatures is the key to develop strategies to understand, treat and diagnose diseases.Decades of research and technological advancements have led to the development of a number of molecular tools and engineering technologies that allow accessing the information contained in the nucleic acids. This thesis provides a general overview of the tools and technologies available for nucleic acid analysis, and proposes an illustrative concept on how molecular tools and emergent technologies can be combined in a modular fashion to design methods for addressing different biomedical questions. The studies included in this thesis, are focused on the particular use of the molecular tools named: padlock and selector probes, rolling circle amplification, and fluorescence detection of single molecules in combination with microfluidics and portable microscopy. By using this combination, it became possible to design and demonstrate novel approaches for integrated nucleic acid analysis, inexpensive digital quantification, mobile-phone based diagnostics and the description of viral infections.These studies represent a step forward towards the adoption of the selected group of tools and technologies, for the design and building of methods that can be used as powerful alternatives to conventional tools used in molecular diagnostics and virology.
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| 9. |
- Hernández-Neuta, Iván, et al.
(författare)
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Smartphone-based clinical diagnostics : towards democratization of evidence-based health care
- 2019
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Ingår i: Journal of Internal Medicine. - : Wiley. - 0954-6820 .- 1365-2796. ; 285:1, s. 19-39
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Forskningsöversikt (refereegranskat)abstract
- Recent advancements in bioanalytical techniques have led to the development of novel and robust diagnostic approaches that hold promise for providing optimal patient treatment, guiding prevention programs and widening the scope of personalized medicine. However, these advanced diagnostic techniques are still complex, expensive and limited to centralized healthcare facilities or research laboratories. This significantly hinders the use of evidence-based diagnostics for resource-limited settings and the primary care, thus creating a gap between healthcare providers and patients, leaving these populations without access to precision and quality medicine. Smartphone-based imaging and sensing platforms are emerging as promising alternatives for bridging this gap and decentralizing diagnostic tests offering practical features such as portability, cost-effectiveness and connectivity. Moreover, towards simplifying and automating bioanalytical techniques, biosensors and lab-on-a-chip technologies have become essential to interface and integrate these assays, bringing together the high precision and sensitivity of diagnostic techniques with the connectivity and computational power of smartphones. Here, we provide an overview of the emerging field of clinical smartphone diagnostics and its contributing technologies, as well as their wide range of areas of application, which span from haematology to digital pathology and rapid infectious disease diagnostics.
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