TY  - JOUR
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
JF  - Microchimica acta : analytical sciences based on micro- and nanomaterials
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 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.
KW  - Isothermal amplification
KW  - RPA
KW  - Microchip
KW  - DNA sensor
KW  - Point-of-care
Y1  - 2014
U6  - https://doi.org/10.1007/s00604-014-1198-5
SN  - 0026-3672
SN  - 1436-5073
VL  - 181
IS  - 13-14
SP  - 1715
EP  - 1723
PB  - Springer
CY  - Wien
ER  -