Enhanced solid-phase recombinase polymerase amplification and electrochemical detection

Abstract

Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

“…7). 68,101,102,[165][166][167] Results suggest that electrochemical detection could be up to 10 5 -fold more sensitive than optical detection (by ELISA). 166 Alternatively, Ng et al 103 and Lau et al 100 RPA in combination with electrochemical detection.…”

“…However, several research groups have studied RPA reaction temperatures that lie outside of the recommended range. 38,44,45,60,[62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78] The largest temperature range was tested between 15°C and 50°C; 62,64,69,70,76 and results indicated the marginal reaction temperature to produce a positive result should be greater than 30°C. [62][63][64]66,67,69,71,74,76,77 However, Sun et al 65 and Poulton and Webster 60 showed that temperature as low as 25°C could still generate a positive signal after RPA amplification and subsequent lateral flow strip detection.…”

Recombinase polymerase amplification: Basics, applications and recent advances

“…Several methods have been developed with solid phase amplification with performances usually inferior to that achieved with solution phase amplification [5,27e30,39] as primer accessibility is more restricted impeding amplification efficiency, and future work will need to focus on strategies to decrease amplification time. Efforts to optimise the surface chemistry of the immobilised primers, exploiting vertical and horizontal spacers to enhance solid phase amplification has been reported [40]. To decrease the reaction time and improve the limit of detection, the surface-immobilised primer can also be introduced in the solution phase in an approach termed hemi-nested asymmetric solid-phase amplification [41e43].…”

In Situ Plant Virus Nucleic Acid Isothermal Amplification Detection on Gold Nanoparticle-Modified Electrodes

“…Label-free optical approaches 18,19 , and enzymatic label-based electrochemical ones with high degree of integration have been reported. 20 However, heat sources are needed for getting detectable signals, which represents an important practical limitation.…”