TY - GEN A1 - Kersting, Sebastian A1 - Rausch, Valentina A1 - Bier, Frank Fabian A1 - von Nickisch-Rosenegk, Markus T1 - Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 730 KW - isothermal amplification KW - RPA KW - microchip KW - DNA sensor KW - point-of-care Y1 - 2019 UR - https://publishup.uni-potsdam.de/frontdoor/index/index/docId/43047 UR - https://nbn-resolving.org/urn:nbn:de:kobv:517-opus4-430479 SN - 1866-8372 IS - 730 SP - 1715 EP - 1723 ER -