| 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. |
- 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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| 3. |
- 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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| 4. |
- Kühnemund, Malte, et al.
(författare)
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Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules
- 2017
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 45:8
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Tidskriftsartikel (refereegranskat)abstract
- Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.
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