Fig. 3. Lateral flow detection of HCV LAMP assay.

a The mechanism of the lateral flow strip¹⁵. Two primers, FLP and BLP, are pre-labelled with Biotin and FITC, respectively. The amplicon resulting from the LAMP reaction, contains both labelled primers as double-stranded DNA represented schematically by two gray lines. It is added onto the sample pad and moves towards the conjugate via capillary action. The streptavidin-labelled red particles bind with the Biotin (from the FLP primer) on the amplicon and together move towards the test line. The anti-FITC antibody (Ab) on the strip captures the amplicon via its FITC label (from the BLP primer) at the test line forming a band. Any unbound red particles move towards the control line where they are captured via the Biotin forming a second band. b The assembly and interpretation of the lateral flow devices. The device consists of the water chamber (1), connecting channels (2), four LAMP reaction chambers (3), channels (4) and lateral flow strips (5). Following the incubation period, two bands indicate a positive reaction, one band indicates a negative and no bands indicate invalid results. P—positive, N—negative, 1 and 2—sample in duplicate. c Analytical sensitivity of the lateral flow method (top panel) compared to gel electrophoresis (middle panel) and fluorescence over time (bottom panel). Serial dilutions of plasmid JFH1 replicon were made based on copy number per reaction (log₁₀). Black lines indicate median with interquartile range (n = 3 biologically independent experiments, each with three technical replicates). The different symbols are for each dilution, to ease visualisation (grey and white disc—4.1, white lozenges—3.8, inverse triangles—3.5, black diamond—3.2, grey circles—2.9 and white squares—2.6 log₁₀ copies/reaction, black disc is negative control—DI water). Statistical analysis for the fluorescence over time was performed using a parametric, one-way ANOVA. The F ratio = 55.56 and the degrees of freedom = 65. ****p ≤ 0.0001, Pos—positive HCV control, Neg—no template control, M—100 bp NEB DNA ladder, C—control line, T—test line. Source data are provided as a Source data file.