Fig. 2: Mechanistic of the TRACE assay.

a–d Signal dynamics of (a) two-step CRISPR/Cas12a, and one-pot (b) CRISPR/Cas12a, (c) CRISPR/Cas12b, and (d) TRACE assays when analyzing MPXV J2L gene standards (0-100 copies/test). Created in BioRender. Dong, Y. (https://BioRender.com/931qrw6). All assays used 400 nM RPA primers, 50 nM RNPs, and 500 nM reporter, while the TRACE assay also included 200 nM single-stranded RNA (ssRNA) blocker of the J2L-specific gRNA. Limits of detection (LoDs) were defined as the lowest J2L concentration yielding an endpoint signal significantly greater than the no-template control (NTC; 0 copy/test) background (mean+3×SD).Results are presented as the mean ± SD (shaded region) of three technical replicates. p values were calculated using the two-tailed Wilcoxon signed-rank test relative to non-template control (NTC). J2L amplicon accumulation at designated time points in RPA and one-pot Cas12b, Cas12a, and TRACE assays, as indicated by representative PAGE images (top) and densitometry results (bottom) normalized to the mean of the 30-min RPA values. Numbers indicate the signal detected vs. the corresponding RPA reaction time points. e Cas12b cis-cleavage efficiency and (f) trans-cleavage activity in CRISPR/Cas12b reactions performed at the indicated temperatures with and without a ssRNA gRNA blocker, as defined by (e) the decrease in dsDNA substrate PAGE band intensity after 2 min (Supplementary Fig. 2), and (f) the maximal trans-cleavage rate (Vmax_Trans), as calculated from the first derivative of the fluorescence–time curve (Supplementary Fig. 3). g, h Electrophoretic mobility shift assays of (g) gRNA/Cas12b RNP and (h) RNP–DNA target complex formation at the indicated Cas12b and RNP concentrations with or without ssRNA gRNA blocker as defined by the decrease in intensity of the free gRNA and the free DNA bands (Supplementary Fig. 5). Created in BioRender. Dong, Y. (https://BioRender.com/3fkvomk). Data reflects mean ± SDs values measured or derived from Image J analyses of three technical replicates. Source data are provided as a Source Data file.