Fig. 6

Evaluation and application of an RPA-CRISPR/Cas12a-based lateral flow assay for point-of-care syphilis detection. (A) Feasibility verification of the RPA-CRISPR/Cas12a-LFA for Treponema pallidum detection (1: Complete reaction system; 2: Without Cas12a; 3: Without crRNA; 4: Without ssDNA reporter; 5: Without target). (B) Optimization of amplification time for RPA-CRISPR/Cas12a-LFA detection of Treponema pallidum (1: 0 min; 2: 10 min; 3: 20 min; 4: 30 min; 5: 40 min). (C) Optimization of Cas12a protein concentration for RPA-CRISPR/Cas12a-LFA detection of Treponema pallidum (1: 25 nM; 2: 50 nM; 3: 100 nM; 4: 200 nM; 5: 250 nM). (D) Optimization of crRNA concentration for RPA-CRISPR/Cas12a-LFA detection of Treponema pallidum (1: 50 nM; 2: 100 nM; 3: 200 nM; 4: 250 nM; 5: 500 nM). (E) Optimization of ssDNA reporter concentration for RPA-CRISPR/Cas12a-LFA detection of Treponema pallidum (1: 100 nM; 2: 200 nM; 3: 250 nM; 4: 400 nM; 5: 500 nM). (F) Sensitivity evaluation of the RPA-CRISPR/Cas12a-LFA for Treponema pallidum detection (1: 1 ng/test; 2: 100 pg/test; 3: 10 pg/test; 4: 1 pg/test; 5: 100 fg/test; 6: 10 fg/test; 7: 1 fg/test; 8: NC). (G) Specificity evaluation of the RPA-CRISPR/Cas12a-LFA for Treponema pallidum detection (1: TP, Treponema pallidum; 2: HIV; 3: HBV; 4: HCV; 5: DENV; 6: NC). (H) Clinical sample validation of the RPA-CRISPR/Cas12a-LFA for Treponema pallidum detection (representative results).