Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens

• Published in: Mikrochimica acta (1966) 2014, Vol.181 (13-14), p.1715-1723
• Main Authors: Kersting, Sebastian, Rausch, Valentina, Bier, Frank F., von Nickisch-Rosenegk, Markus
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; DNA; Methicillin; Microengineering; Nanochemistry; Nanotechnology; Original Paper; Pathogenic microorganisms; Staphylococcus aureus
• ISSN: 0026-3672; 1436-5073
• DOI: 10.1007/s00604-014-1198-5

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