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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 degrees 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 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.…
Author details: | Sebastian KerstingGND, Valentina RauschORCiD, Frank Fabian BierORCiDGND, Markus von Nickisch-RosenegkORCiD |
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DOI: | https://doi.org/10.1007/s00604-014-1198-5 |
ISSN: | 0026-3672 |
ISSN: | 1436-5073 |
Title of parent work (English): | Microchimica acta : analytical sciences based on micro- and nanomaterials |
Publisher: | Springer |
Place of publishing: | Wien |
Publication type: | Article |
Language: | English |
Date of first publication: | 2014/02/18 |
Publication year: | 2014 |
Release date: | 2017/03/27 |
Tag: | DNA sensor; Isothermal amplification; Microchip; Point-of-care; RPA |
Volume: | 181 |
Issue: | 13-14 |
Number of pages: | 9 |
First page: | 1715 |
Last Page: | 1723 |
Funding institution: | German Federal Ministry of Education and Research [03IS2201A, 03IS2201B] |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
DDC classification: | 5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften |
Peer review: | Referiert |
Publishing method: | Open Access |
License (German): | CC-BY - Namensnennung 4.0 International |
External remark: | Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 730 |