| 1. |
- 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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| 2. |
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| 3. |
- Engström, Anna, et al.
(författare)
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Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead- Based Readout
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:4, s. e62015-
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Tidskriftsartikel (refereegranskat)abstract
- Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlockprobes were designed to target the most common mutations associated with rifampicinresistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. Fordetection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identificationof the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugatedmagnetic nanobeads and detected by measuring the frequency-dependent magneticresponse of the beads using a portable AC susceptometer.
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| 4. |
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| 5. |
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| 6. |
- 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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| 7. |
- 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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| 8. |
- Mezger, Anja, et al.
(författare)
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A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections
- 2015
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 53:2, s. 425-432
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Tidskriftsartikel (refereegranskat)abstract
- To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection.
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| 9. |
- Mezger, Anja, et al.
(författare)
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Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:11, s. e111874-
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Tidskriftsartikel (refereegranskat)abstract
- Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.
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| 10. |
- Mezger, Anja, et al.
(författare)
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Highly specific DNA detection employing ligation on suspension bead array readout
- 2015
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 32:5, s. 504-510
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Tidskriftsartikel (refereegranskat)abstract
- We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without the need of PCR amplification. Furthermore, using monomerized RCA products to guide ligation of the detection oligonucleotide (DO) to barcode sequences on the magnetic Luminex beads, combined with efficient washing and increased measurement temperature, yields a higher signal to noise ratio. As a proof-of-principle, we demonstrate detection of pathogenic DNA sequences with high reproducibility, sensitivity and a dynamic range over four orders of magnitude. Using padlock probes in combination with bead suspension arrays opens up the possibility for highly multiplexed DNA targeting and readout.
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