Figure 1

Recombinase polymerase amplification and lateral flow detection. Reaction principle of the recombinase polymerase amplification (RPA). The two oligonucleotide primers form a complex with the recombinase proteins (green). This complex is able invade the target DNA and directs the primer to homologous sequences. A continuous amplification at 38°C takes place by strand-displacement synthesis catalyzed by a DNA polymerase (red) while single-strand binding proteins (SSB) (brown) stabilize the displaced strand (A). During LF-RPA the reverse primer carries an antigenic tag at the 5′end and a modified probe is added in the reaction. The modified probe is labelled with a different antigenic tag at the 5′end and a 3′end polymerase blocking group. Additionally the probe contains an abasic site 30 nt downstream the 5′end. Only when the probe fully binds to a homologous sequence the nuclease (yellow) is able to cut through the DNA double strand and release the blocking group. Thereby additional polymerase extension substrate is generated and the continuation of strand synthesis results in a dual-labelled amplicon. Amplification and labelling reaction run in the same tube (B). Detection of the labelled RPA amplicon on the lateral flow dipstick by binding to tag-specific antibodies on the test strip and to tag-specific antibodies on gold nanoparticles present in the sample application area. A reddish band is generated on the test line in a sandwich assay manner if the amplicon was successfully generated. Not captured gold particles flow over and are fixed by species-specific antibodies on the control line (C). LF-RPA for P. falciparum using 1 ng of genomic DNA and 25 min reaction time. A test band visible to the naked eye is formed and confirms the presence of the RPA amplicon. The control line determines the validity of the test run. Detail enhanced for better view (D).