Fig. 5: Evaluation of the VaNGuard test under more realistic conditions.

a 20,000 copies of synthetic SARS-CoV-2 RNA fragments were spiked into 10 ng of total RNA extracted from different immortalized human cell lines to mimic infection in various cell types [HEK293T: adrenal precursor, A549: lung, PC9: lung, HCC2279: lung, HL60: blood (promyelocytes), THP-1: blood (monocytes), U937: blood (monocytes), K562: blood (granulocytes/ erythrocytes), and Jurkat: blood (T cells)]. Pure synthetic viral RNAs were used as a control. The control or spiked RNA samples served as input to RT-LAMP, which was performed at 65 °C for 15 min. The Cas detection reaction was then carried out at 37 °C, with fluorescence after 30 min shown. Data represent mean ± s.e.m. (n = 3 [all except control and HEK293T RNA], 6 [control], or 7 [HEK293T RNA] biological replicates). There was no significant loss of signal in the presence of human RNA (n.s.: not significant, P > 0.2; one-sided Student’s t-test). b Analytical LoD for a S254F N234N double mutant RNA template either by itself or mixed with 10 ng total human RNA from HCC2279 cells. Different copies of synthetic viral template (with or without human 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 37 °C, with fluorescence after 10 min shown. Data represent mean ± s.e.m. (n = 4 biological replicates). c Analytical LoD for purified synthetic wild-type SARS-CoV-2 RNA template in the presence of 2 µl or 4 µl UTM. Data represent mean ± s.e.m. (n = 4 biological replicates). d Analytical LoD for wild-type (WT) or S254F N234N double mutant RNA template mixed with 10 ng total human RNA from HCC2279 cells in the presence of 2 µl UTM. Data represent mean ± s.e.m. (n = 4 biological replicates). Source data are available in the Source Data file.