Fig. 4: CreDiT probe design.
a For a given target DNA (gray backbone), a pair of CreDiT probes (forward and reverse) were designed, with each probe consisting of an RPA primer (green backbone) and a Cas12a/gRNA complex (orange backbone). Upon binding to the target DNA, the primer exposes a downstream sequence complementary to gRNA. The Cas12a/gRNA complex then recognizes its target and becomes an active endonuclease to cleave F-Q reporters. HPV16 probes are shown. b Assay validation with varying reaction conditions. HPV16 DNA was used as a model target ([DNA] = 4 × 107 copies/mL). A high signal was only observed when all assay components were present. Data were displayed as mean ± s.d. from technical triplicates. c CreDiT (20 min) and qPCR (1 h) assays were used to analyze samples with different target DNA concentrations. The CReDiT assay exhibited a lower detection limit (40 copies/mL) and a wider dynamic range (4 orders of magnitude) than qPCR (detection limit, 400 copies/mL; dynamic range, 2.5 orders of magnitude). ∆CreDiT is the background-subtracted signal. Data were displayed as mean ± s.d. from technical triplicates. d Probe selectivity. A panel of probes was designed for hrHPV targets (HPV16, HPV18, HPV31, HPV33, HPV45, and HPV58) and GAPDH. These probes detected their intended targets with negligible off-target signals. [Target DNA] = 4 × 107 copies/mL. The heatmap displays mean values from technical triplicates. a.u. arbitrary unit. Source data are provided as a Source Data file.
