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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,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.show moreshow less

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Metadaten
Author details:Sebastian KerstingGND, Valentina RauschORCiD, Frank Fabian BierORCiDGND, Markus von Nickisch-RosenegkORCiD
URN:urn:nbn:de:kobv:517-opus4-430479
DOI:https://doi.org/10.25932/publishup-43047
ISSN:1866-8372
Title of parent work (German):Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe
Publication series (Volume number):Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe (730)
Publication type:Postprint
Language:English
Date of first publication:2019/06/21
Publication year:2014
Publishing institution:Universität Potsdam
Release date:2019/06/21
Tag:DNA sensor; RPA; isothermal amplification; microchip; point-of-care
Issue:730
Number of pages:9
First page:1715
Last Page:1723
Source:Microchimica Acta 181 (2014) 13–14, S. 1715–1723 DOI: 10.1007/s00604-014-1198-5
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät
DDC classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
Peer review:Referiert
Publishing method:Open Access
License (German):License LogoCC-BY - Namensnennung 4.0 International
External remark:Bibliographieeintrag der Originalveröffentlichung/Quelle
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