Fig. 10: Development of a human internal control for our VaNGuard test.

a Strip chart showing the efficacy of different sets of LAMP primers targeting the human POP7, ACTB, or GAPDH gene. The primers labelled with “Set1”, “Set2”, or “Set3” are newly designed, while the primers labelled with “Pub” have been published^17,72,73. 2 µl heat-treated saliva was used as sample input to RT-LAMP, which was performed at 65 °C over 40 min in a real-time instrument. The black horizontal bars among the data points in the strip chart represent the mean (n = 6 [POP7 Pub and Set1-2, ACTB Set2-3, GAPDH Set2-3], 8 [ACTB Set1, GAPDH Pub and Set1], or 14 [ACTB Pub] biological replicates). Comparisons were done relative to the POP7 Pub primers¹⁷. P-values were calculated using one-sided Student’s t-test. b Strip chart showing the effect of different human primer sets on isothermal amplification of the SARS-CoV-2 S-gene. Different copies of synthetic SARS-CoV-2 RNA were used as sample input to RT-LAMP, which was performed at 65 °C over 40 min in a real-time instrument. The black horizontal bars among the data points in the strip chart represent the mean (n = 3 [POP7 Pub 2E5] or 6 [POP7 Pub all except 2E5, ACTB Set2] biological replicates). c, d Evaluation of our prototype VaNGuard assay containing a human internal control using c clinically negative and d clinically positive NP swab samples. The green fluorescence originates from a generic DNA-binding dye, while the red fluorescence originates from a Cy5-quencher reporter specific for SARS-CoV-2. Each sample was treated with proteinase K and heat before 2 µl was used as input to the quasi-one-pot reaction. e 20 copies of synthetic SARS-CoV-2 RNA were used as input to the quasi-one-pot reaction with different amounts of pyrophosphatase added during the Cas detection step. The fluorescence measurements here were taken after 5 min of trans-cleavage reaction. Data represent mean ± s.e.m. (n = 4 biological replicates). P-value was calculated using one-sided Student’s t-test. f Evaluation of our assay with various amounts of human primers and pyrophosphatase. Different copies of synthetic RNA spiked into heat-treated saliva 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). g Clinical evaluation of our optimized VaNGuard assay containing a human internal control. 2 µl of each proteinase K- and heat-treated NP swab sample was used as input. Source data are available in the Source Data file.