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

Sebastian Kersting,Markus Von Nickisch-Rosenegk,Valentina Rausch,Frank F Bier

doi:10.1007/s00604-014-1198-5

Abstract

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. FigureThe combination of multiplex isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides Electronic supplementary materialThe online version of this article (doi:10.1007/s00604-014-1198-5) contains supplementary material, which is available to authorized users.

Highlights

The amplification of a specific DNA sequence for the detection of a pathogen is a common tool in molecular diagnostics
We tested the speed of the on-chip recombinase polymerase amplification (RPA) with 1 ng of genomic DNA of Salmonella enterica and stopped the reaction at 0, 5, 10, 20 and 30 min by starting the washing process
We described the combination of isothermal nucleic acid amplification with RPA and detection on immobilized probes

Summary

Introduction

The amplification of a specific DNA sequence for the detection of a pathogen is a common tool in molecular diagnostics. Examples for isothermal nucleic acid amplification techniques are the loop mediated amplification (LAMP) [5], the helicase dependent amplification (HDA) [6] the strand-displacement amplification (SDA) [7] and the rolling circle amplification (RCA) [8] which have been recently described and have already proven their use [9, 10]. Another promising method is the recombinase polymerase amplification (RPA) which uses a phage recombinase to direct short oligonucleotide primers to a homologous target sequence. Few applications of this method have been integrated in more complex instrumentation as an approach for an improved nucleic acid diagnosis in point of care testing [17,18,19,20,21]

Results

Discussion

Conclusion