Fig. 1
From: Detection of P. malariae using a new rapid isothermal amplification lateral flow assay
Plasmodium malariae lateral-flow based RPA assay schematic. (1) Reaction components include unlabelled forward and 5′-biotinylated reverse primers, 5′-FAM labelled probe with an abasic residue and 3′ blocker, endonuclease IV enzyme (nfo), and template DNA. (2) RPA reaction proceeds via primer-recombinase-SSB complex de-looping, nfo cleavage, polymerase extension that results in FAM- and biotin-labelled double-stranded amplicons. Exponential amplification occurs during 20-min isothermal incubation at 39 °C. (3) Lateral flow strips with a band containing anti-FAM antibodies are loaded with RPA product diluted in buffer containing streptavidin-conjugated gold nanoparticles. (4) Detection of labelled RPA product immobilized on the lateral flow strip is performed by visual inspection using the naked eye. Total reaction time is approximately 35 min. Figure made using BioRender