| 1. |
- Barisic, Ivan, et al.
(författare)
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Multiplex detection of antibiotic resistance genes using padlock probes
- 2013
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Ingår i: Diagnostic microbiology and infectious disease. - : Elsevier BV. - 0732-8893 .- 1879-0070. ; 77:2, s. 118-125
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Tidskriftsartikel (refereegranskat)abstract
- The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse beta-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the beta-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10(4) DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of beta-lactamase genes in dilutions of 10(7) cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.
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| 2. |
- Göransson, Jenny, et al.
(författare)
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Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2, s. e31068-
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Tidskriftsartikel (refereegranskat)abstract
- Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.
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| 3. |
- Ke, Rongqin, et al.
(författare)
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Colorimetric Nucleic Acid Testing Assay for RNA Virus Detection Based on Circle-to-Circle Amplification of Padlock Probes
- 2011
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 49:12, s. 4279-4285
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Tidskriftsartikel (refereegranskat)abstract
- We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 103 viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.
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| 4. |
- Ke, Rongqin, et al.
(författare)
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Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7
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Tidskriftsartikel (refereegranskat)abstract
- Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.
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| 5. |
- Ke, Rongqin, et al.
(författare)
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In situ sequencing for RNA analysis in preserved tissue and cells
- 2013
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 10:9, s. 857-860
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Tidskriftsartikel (refereegranskat)abstract
- Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue. We demonstrate in situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections.
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| 6. |
- Sun, Song, et al.
(författare)
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Genome-Wide Detection of Spontaneous Chromosomal Rearrangements in Bacteria
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:8, s. e42639-
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Tidskriftsartikel (refereegranskat)abstract
- Genome rearrangements have important effects on bacterial phenotypes and influence the evolution of bacterial genomes. Conventional strategies for characterizing rearrangements in bacterial genomes rely on comparisons of sequenced genomes from related species. However, the spectra of spontaneous rearrangements in supposedly homogenous and clonal bacterial populations are still poorly characterized. Here we used 454 pyrosequencing technology and a 'split mapping' computational method to identify unique junction sequences caused by spontaneous genome rearrangements in chemostat cultures of Salmonella enterica Var. Typhimurium LT2. We confirmed 22 unique junction sequences with a junction microhomology more than 10 bp and this led to an estimation of 51 true junction sequences, of which 28, 12 and 11 were likely to be formed by deletion, duplication and inversion events, respectively. All experimentally confirmed rearrangements had short inverted (inversions) or direct (deletions and duplications) homologous repeat sequences at the endpoints. This study demonstrates the feasibility of genome wide characterization of spontaneous genome rearrangements in bacteria and the very high steady-state frequency (20-40%) of rearrangements in bacterial populations.
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| 7. |
- Zardán Gómez de la Torre, Teresa, et al.
(författare)
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Sensitive Detection of Spores Using Volume-Amplified Magnetic Nanobeads
- 2012
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Ingår i: Small. - : Wiley. - 1613-6810. ; 8:14, s. 2174-2177
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Tidskriftsartikel (refereegranskat)abstract
- A magnetic-nanobead-based, substrate-free method for the sensitive detection of spores in an immunoassay format is presented. The method is shown to detect Bacillus globigii spores, the non-pathogenic simulant of Bacillus anthracis, with a limit-of-detection of 50 spores with a reaction time of 135 min. The study shows the versatility of magnetic nanobeads for detection of biological molecules other than DNA.
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| 8. |
- Zhao, Yansong, et al.
(författare)
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Single Cell RNA Expression Analysis Using Flow Cytometry Based on Specific Probe Ligation and Rolling Circle Amplification
- 2020
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Ingår i: ACS Sensors. - : American Chemical Society (ACS). - 2379-3694. ; 5:10, s. 3031-3036
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Tidskriftsartikel (refereegranskat)abstract
- Conventional flow cytometry has been widely used for high-throughput single-cell gene expression analysis using specific antibody staining. However, this is limited by the availability of high-quality antibodies. We developed a novel flow cytometry RNA detection technique termed RCA-Flow for single-cell RNA expression analysis. We showed that it is able to analyze not only mRNAs but also microRNAs and circular RNAs that are otherwise difficult to analyze by other flow cytometry techniques. The versatility for high-throughput analysis of different types of RNA molecules makes our method possess great potential for both biomedical and clinical applications.
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