Fig. 6: Modification of ZBP RT-LAMP assay for detection of SARS-CoV-2 from crude gargle samples.

a Heat map depicting the optimization of heat protocol and components of 10 × inactivation buffer. The conditions highlighted in yellow depict the optimal parameter that was carried over to the next testing phase; therefore, modifications are additive going from top to bottom. b Comparison of amplification characterstics between reactions run with gargle versus 10 mM citrate buffer (pH = 6) as input. Both sample types first underwent the full inactivation procedure before being spiked with the same amount of RNA. c Colorimetric and fluorescent performance of RT-LAMP with different volumes of gargle spiked with 10 copies of viral RNA per µl of gargle compared to 30 copies of viral RNA in 10 mM citrate (pH = 6) used as reference. Identical gargle volume was used in NTC reactions. d Scheme demonstrating the workflow for LAMP based detection of SARS-CoV-2. Gargle collection is followed by the inactivation, heat-treatment, and short centrifugation to exclude proteineous precipitates. Inactivated samples are then added to prepared master mix, incubated for 45 min at 65 °C, and analyzed by visual confirmation by the naked eye. e Detection limit of Direct ZBP RT-LAMP assay using a mock clinical gargle sample. Depicted Ct of the neat sample that was determined by RT-qPCR and served as the reference point for serial fold-dilution. Exemplary melt curve of LAMP products is shown as an inset. The highest time to reaction value on y-axis equals the total duration of the reaction. Amplification success rate represents the percentage of samples that amplified over the course of the reaction. Gargle without viral RNA was used in NTC reactions. All experiments were performed using “ZBP 1.1 G” reaction mix. Error bars represent standard error of the mean. In all experiments, n = 8 technical replicates per group were used. NTC no template control, TTR time to reaction, TCEP Tris(2-carboxyethyl)phosphine, cp/µl copies per microliter.