43641
2017
2018
eng
215
doctoralthesis
1
--
--
2018-06-25
Isothermal nucleic acid amplification for the detection of infectious pathogens
Dissertation, Universiät Potsdam, 2018
false
true
Sebastian Kersting
Biowissenschaften; Biologie
Institut für Biochemie und Biologie
37995
2014
2014
eng
9
13
article
BioMed Central
London
1
--
--
--
Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis
Background: Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing.
Methods: A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38 C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay.
Results: The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45 degrees C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined.
Conclusions: Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite.
Malaria journal
10.1186/1475-2875-13-99
24629133
1475-2875
wos:2014
99
WOS:000334803200002
Kersting, S (reprint author), Fraunhofer Inst Biomed Engn IBMT, Branch Potsdam, Dept Nanobiotechnol & Nanomed, Muehlenberg 13, D-14476 Potsdam, Germany., sebastian.kersting@ibmt.fraunhofer.de
German Federal Ministry of Education and Research [03IS2201A, 03IS2201B]
<a href="https://doi.org/10.25932/publishup-43156">Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 948</a>
Sebastian Kersting
Valentina Rausch
Frank Fabian Bier
Markus von Nickisch-Rosenegk
eng
uncontrolled
Plasmodium falciparum
eng
uncontrolled
Recombinase polymerase amplification
eng
uncontrolled
RPA
eng
uncontrolled
PCR
eng
uncontrolled
Lateral flow
eng
uncontrolled
Point-of-care testing
eng
uncontrolled
Rapid test
eng
uncontrolled
Isothermal nucleic acid amplification
Institut für Biochemie und Biologie
Referiert
Open Access
37504
2014
2014
eng
1715
1723
9
13-14
181
article
Springer
Wien
1
2014-02-18
2014-02-18
--
Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens
We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 degrees C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in < 20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics.
Microchimica acta : analytical sciences based on micro- and nanomaterials
10.1007/s00604-014-1198-5
0026-3672
1436-5073
wos:2014
WOS:000344001000026
Kersting, S (reprint author), Fraunhofer Inst Biomed Engn IBMT, Branch Potsdam, Muehlenberg 13, D-14476 Potsdam, Germany., sebastian.kersting@ibmt.fraunhofer.de; frank.bier@ibmt.fraunhofer.de
German Federal Ministry of Education and Research [03IS2201A, 03IS2201B]
<a href="http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-430479">Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 730</a>
CC-BY - Namensnennung 4.0 International
Sebastian Kersting
Valentina Rausch
Frank Fabian Bier
Markus von Nickisch-Rosenegk
eng
uncontrolled
Isothermal amplification
eng
uncontrolled
RPA
eng
uncontrolled
Microchip
eng
uncontrolled
DNA sensor
eng
uncontrolled
Point-of-care
Chemie und zugeordnete Wissenschaften
open_access
Institut für Biochemie und Biologie
Referiert
Open Access
52832
2018
2018
eng
54
60
7
550
article
Elsevier
San Diego
1
2018-04-18
2018-04-18
--
A recombinase polymerase amplification assay for the diagnosis of atypical pneumonia
Pneumonia is one of the most common and potentially lethal infectious conditions worldwide. Streptococcus pneumoniae is the pathogen most frequently associated with bacterial community-acquired pneumonia, while Legionella pneumophila is the major cause for local outbreaks of legionellosis. Both pathogens can be difficult to diagnose since signs and symptoms are nonspecific and do not differ from other causes of pneumonia. Therefore, a rapid diagnosis within a clinically relevant time is essential for a fast onset of the proper treatment. Although methods based on polymerase chain reaction significantly improved the identification of pathogens, they are difficult to conduct and need specialized equipment. We describe a rapid and sensitive test using isothermal recombinase polymerase amplification and detection on a disposable test strip. This method does not require any special instrumentation and can be performed in less than 20 min. The analytical sensitivity in the multiplex assay amplifying specific regions of S. pneumoniae and L. pneumophila simultaneously was 10 CFUs of genomic DNA per reaction. In cross detection studies with closely related strains and other bacterial agents the specificity of the RPA was confirmed. The presented method is applicable for near patient and field testing with a rather simple routine and the possibility for a read out with the naked eye.
Analytical biochemistry : methods in the biological sciences
10.1016/j.ab.2018.04.014
29678761
0003-2697
1096-0309
wos:2018
WOS:000436056600008
Kersting, S (reprint author), Fraunhofer IZI BB, Branch Bioanalyt & Bioproc, Fraunhofer Inst Cell Therapy & Immunol, Muehlenberg 13, D-14476 Potsdam, Germany., sebastian.kersting@izi-bb.fraunhofer.de
German Federal Ministry of Education and ResearchFederal Ministry of Education & Research (BMBF) [03IS2201A, 03IS2201B]
2021-11-25T08:58:55+00:00
sword
importub
filename=package.tar
b65139d35b77a24eac21168d9c1fbea0
Kersting, Sebastian
false
true
Sebastian Kersting
Valentina Rausch
Frank Fabian Bier
Markus von Nickisch-Rosenegk
Biowissenschaften; Biologie
Institut für Biochemie und Biologie
Referiert
Import
43047
2014
2019
eng
1715
1723
9
730
postprint
1
2019-06-21
2019-06-21
--
Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens
We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in <20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics.
Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe
10.25932/publishup-43047
urn:nbn:de:kobv:517-opus4-430479
1866-8372
online registration
Microchimica Acta 181 (2014) 13–14, S. 1715–1723 DOI: 10.1007/s00604-014-1198-5
<a href="http://publishup.uni-potsdam.de/opus4-ubp/frontdoor/index/index/docId/37504">Bibliographieeintrag der Originalveröffentlichung/Quelle</a>
false
true
CC-BY - Namensnennung 4.0 International
Sebastian Kersting
Valentina Rausch
Frank Fabian Bier
Markus von Nickisch-Rosenegk
Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
730
eng
uncontrolled
isothermal amplification
eng
uncontrolled
RPA
eng
uncontrolled
microchip
eng
uncontrolled
DNA sensor
eng
uncontrolled
point-of-care
Chemie und zugeordnete Wissenschaften
open_access
Mathematisch-Naturwissenschaftliche Fakultät
Referiert
Open Access
Universität Potsdam
https://publishup.uni-potsdam.de/files/43047/pmnr730.pdf
43156
2014
2020
eng
11
948
postprint
1
2020-06-05
2020-06-05
--
Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis
Background
Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing.
Methods
A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay.
Results
The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined.
Conclusions
Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite.
Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
10.25932/publishup-43156
urn:nbn:de:kobv:517-opus4-431562
1866-8372
Malaria Journal 13 (2014) 99 DOI: 10.1186/1475-2875-13-99
99
<a href="http://publishup.uni-potsdam.de/opus4-ubp/frontdoor/index/index/docId/37995">Bibliographieeintrag der Originalveröffentlichung/Quelle</a>
Sebastian Kersting
Valentina Rausch
Frank Fabian Bier
Markus von Nickisch-Rosenegk
Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
948
eng
uncontrolled
Plasmodium falciparum
eng
uncontrolled
Recombinase polymerase amplification
eng
uncontrolled
RPA
eng
uncontrolled
PCR
eng
uncontrolled
lateral flow
eng
uncontrolled
point-of-care testing
eng
uncontrolled
rapid test
eng
uncontrolled
isothermal nucleic acid amplification
Medizin und Gesundheit
open_access
Mathematisch-Naturwissenschaftliche Fakultät
Referiert
Open Access
Universität Potsdam
https://publishup.uni-potsdam.de/files/43156/pmnr948.pdf
58562
2020
2020
eng
3445
3453
9
11
83
article
American Chemical Society
Washington
1
2020-11-10
2020-11-10
--
Total syntheses of prenylated isoflavones from Erythrina sacleuxii and their antibacterial activity
The prenylated isoflavones 5-deoxyprenylbiochanin A (7-hydroxy-4'-methoxy-3'-prenylisoflavone) and erysubin F (7,4'-dihydroxy-8,3'-diprenylisoflavone) were synthesized for the first time, starting from mono-or di-O-allylated chalcones, and the structure of 5-deoxy-3'-prenylbiochanin A was corroborated by single-crystal X-ray diffraction analysis. Flavanones are key intermediates in the synthesis. Their reaction with hypervalent iodine reagents affords isoflavones via a 2,3-oxidative rearrangement and the corresponding flavone isomers via 2,3-dehydrogenation. This enabled a synthesis of 7,4'-dihydroxy-8,3'-diprenylflavone, a non-natural regioisomer of erysubin F. Erysubin F (8), 7,4'-dihydroxy-8,3'-diprenylflavone (27), and 5-deoxy-3'prenylbiochanin A (7) were tested against three bacterial strains and one fungal pathogen. All three compounds are inactive against Salmonella enterica subsp. enterica (NCTC 13349), Escherichia coli (ATCC 25922), and Candida albicans (ATCC 90028), with MIC values greater than 80.0 mu M. The diprenylated natural product erysubin F (8) and its flavone isomer 7,4'-dihydroxy-8,3'diprenylflavone (27) show in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) at MIC values of 15.4 and 20.5 mu M, respectively. In contrast, the monoprenylated 5-deoxy-3'-prenylbiochanin A (7) is inactive against this MRSA strain.
Journal of natural products
5-deoxy-3′-prenylbiochanin A and erysubin F
10.1021/acs.jnatprod.0c00932
33170684
0163-3864
1520-6025
outputup:dataSource:WoS:2020
WOS:000595546200027
Schmidt, B (corresponding author), Univ Potsdam, Inst Fuer Chem, D-14476 Potsdam, Germany., bernd.schmidt@uni-potsdam.de
transnational E-RARE grant `CCMCURE (DFG)European Commission [SFB958]; E-RARE [ERL 138397]; Canadian; Institutes for Health ResearchCanadian Institutes of Health Research; (CIHR) [PJT 153000]; the E-RARE grant `CCMCURE
Schmidt, Bernd
2023-03-28T04:13:55+00:00
sword
importub
filename=package.tar
389a2d8863863d3fffdde7ec51ed9490
1491522-4
304325-3
false
true
George Kwesiga
Alexandra Kelling
Sebastian Kersting
Eric Sperlich
Markus von Nickisch-Rosenegk
Bernd Schmidt
Chemie und zugeordnete Wissenschaften
Institut für Chemie
Referiert
Import
59403
2020
2020
eng
30052
30065
14
27
12
article
American Chemical Society
Washington
1
2020-06-09
2020-06-09
--
Impact of multivalence and self-assembly in the design of polymeric antimicrobial peptide mimics
Antimicrobial resistance is an increasingly serious challenge for public health and could result in dramatic negative consequences for the health care sector during the next decades. To solve this problem, antibacterial materials that are unsusceptible toward the development of bacterial resistance are a promising branch of research. In this work, a new type of polymeric antimicrobial peptide mimic featuring a bottlebrush architecture is developed, using a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and ring-opening metathesis polymerization (ROMP). This approach enables multivalent presentation of antimicrobial subunits resulting in improved bioactivity and an increased hemocompatibility, boosting the selectivity of these materials for bacterial cells. Direct probing of membrane integrity of treated bacteria revealed highly potent membrane disruption caused by bottlebrush copolymers. Multivalent bottlebrush copolymers clearly outperformed their linear equivalents regarding bioactivity and selectivity. The effect of segmentation of cationic and hydrophobic subunits within bottle brushes was probed using heterograft copolymers. These materials were found to self-assemble under physiological conditions, which reduced their antibacterial activity, highlighting the importance of precise structural control for such applications. To the best of our knowledge, this is the first example to demonstrate the positive impact of multivalence, generated by a bottlebrush topology in polymeric antimicrobial peptide mimics, making these polymers a highly promising material platform for the design of new bactericidal systems.
ACS applied materials & interfaces
10.1021/acsami.0c05944
32517467
1944-8244
1944-8252
outputup:dataSource:WoS:2020
WOS:000550633400004
Hartlieb, M (corresponding author), Univ Potsdam, Inst Chem, D-14476 Potsdam, Germany.; Hartlieb, M (corresponding author), Fraunhofer Inst Appl Polymer Res IAP, Dept Life Sci & Bioproc, D-14476 Potsdam, Germany., mhartlieb@unipotsdam.de
Open-Topic Postdoc program of the University of Potsdam
Hartlieb, Matthias
2023-06-02T07:05:22+00:00
sword
importub
filename=package.tar
0aa0a5e06219927d44569b04269d1454
2467494-1
false
true
Sophie Laroque
Martin Reifarth
Marcel Sperling
Sebastian Kersting
Stefanie Kloepzig
Patrick Budach
Matthias Hartlieb
Joachim Storsberg
eng
uncontrolled
RAFT polymerization
eng
uncontrolled
ROMP
eng
uncontrolled
antimicrobial polymers
eng
uncontrolled
antimicrobial peptide
eng
uncontrolled
mimics
eng
uncontrolled
bottlebrush copolymers
Chemie und zugeordnete Wissenschaften
Institut für Chemie
Referiert
Import