Fig. 3.
The dWS-CRISPR assay for SARS-CoV-2 detection. (a) A typical workflow of dWS-CRISPR assay to detect SARS-CoV-2 in clinical samples. (b) Endpoint fluorescence micrographs of the QuantStudio digital chip for the SARS-CoV-2 detection with various incubation time (0, 10, 30, 60, 90 and 120 min) at 52 °C. In this dWS-CRISPR assay, 1 × 106 copies/μl SARS-CoV-2 RNA was loaded. (c) The percentage of positive spots comparison for the dWS-CRISPR assays with various incubation time at 52 °C.The number of positive spots was counted by setting the same threshold in the ImageJ software. Percentages of positive spots in each micrograph was calculated (n = 6). Error bars represent the means ± s.d. from replicates. The statistical significance was analyzed using unpaired two-tailed t-test. (d) Effect of various waiting time at room temperature on dWS-CRISPR assay and digital RT-AIOD-CRISPR assay during reaction solution preparation and distribution steps. After specific waiting time at room temperature, the chips were directly observed without incubation. Positive, the reaction with 5 × 105 copies/μl SARS-CoV-2 RNA. NTC, non-template control. Scale bars are 300 μm. Each micrograph is a representative of six distinct regions taken to cover about 2809 microreactions.