Fig. 8: Implementation of a quasi-one-pot reaction.

a Analytical LoD based on the original workflow. Different copies of synthetic SARS-CoV-2 RNA were used as input to RT-LAMP, which was performed at 65 °C for 15 min. The cleavage reaction was then carried out on only 4 µl LAMP products at 60 °C, with fluorescence measurements here taken after 10 min using a microplate reader. Data represent mean ± s.e.m. (n = 3 biological replicates). b Analytical LoD when all LAMP products were utilized. After RT-LAMP was completed, 50 µl CRISPR reagents were added directly into each sample tube for the cleavage reaction. Data represent mean ± s.e.m. (n = 3 biological replicates). c Strip chart showing how LAMP sensitivity was altered by substituting 0.1 M glycine (Gly) with 40 mM guanidine (Gua). RT-LAMP was performed at 65 °C in a real-time instrument with variable copies of synthetic RNA. The black horizontal bars among the data points in the strip chart represent the mean (n = 5 biological replicates). P-values were calculated using one-sided Student’s t-test. d Comparing the assay sensitivity between glycine and guanidine. Different copies of synthetic SARS-CoV-2 RNA were used as input to RT-LAMP, which was performed at 65 °C for 15 min. Subsequently, 50 µl CRISPR reagents were added directly into each sample tube and the cleavage reaction was carried out at 60 °C, with fluorescence measurements here taken after 10 min using a microplate reader. Data represent mean ± s.e.m. (n = 8 biological replicates). e Lateral flow assays to assess cleavage reaction kinetics with guanidine in the assay mix. The enAsCas12a enzyme was complexed with both the S2 hybrid guide and the S6 5’-extended gRNA. After RT-LAMP was completed, the Cas detection reaction was performed at 60 °C for 5, 7, or 10 min before a dipstick was added to each sample tube. f Lateral flow assays to evaluate VaNGuard test sensitivity with guanidine in the assay mix. Different copies of synthetic SARS-CoV-2 RNA were used as input to RT-LAMP, which was performed at 65 °C for 15 min. The Cas detection reaction was then carried out at 60 °C for 5 min before a dipstick was added to each sample tube. g Analytical LoD for WT or S254F N234N double mutant RNA template using a quasi-one-pot reaction. The enAsCas12a enzyme was complexed with both the S2 and S6 hybrid guides. Fluorescence measurements here were taken after 5 min of trans-cleavage reaction. Data represent mean ± s.e.m. (n = 3 biological replicates). h Evaluating the specificity of our VaNGuard test. The enAsCas12a enzyme was complexed with both the S2 and S6 hybrid guides. 1E6 copies of synthetic RNA from different respiratory viruses were used as input to the quasi-one-pot reaction. Fluorescence measurements were taken at 5-min intervals using a microplate reader. Data represent mean ± s.e.m. (n = 3 biological replicates). i Evaluation with clinical RNA samples. Ct values were obtained using the Fortitude Kit. The enAsCas12a enzyme was complexed with both the S2 and S6 hybrid guides. 2 µl of each RNA sample was used as input to the quasi-one-pot reaction. The Cas detection reaction was performed for 5 min before a dipstick was added to each sample tube. A ratio of less than 0.15 was considered to be negative in our test. Hence, for the purified RNA samples, the lateral flow assay gave 0 false positives and 8 false negatives (RP6, RP45-51). j Strip chart summarizing the results from the clinical evaluation of our VaNGuard test using purified RNA samples. “Yes” indicates that the samples emerged positive in our test, while “No” indicates that the samples emerged negative. Source data are available in the Source Data file.