Fig. 3: Optimization of extraction-free viral lysis.

The real-time fluorescence curves and their end-point fluorescence intensity of the 10-minutes single-step CRISPR reaction using the lysed (a) rash fluid swab, (b) oral swab, (c) saliva and (d) urine samples with different lysis temperature (room temperature, 40, 80, 95, and 98 °C). Lysis time: 5 minutes. The real-time fluorescence curves and their end-point fluorescence intensity of the 10-minutes single-step CRISPR reaction using the lysed (e) rash fluid swab, (f) oral swab, (g) saliva and (h) urine samples with different lysis time (0, 2, 5, 10, and 15 minutes). Lysis temperature: 80 °C. The real-time fluorescence curves of the 10-minutes single-step CRISPR reaction using the lysed (i) rash fluid swab, (j) oral swab, (k) saliva and (l) urine samples and their (m) end-point fluorescence intensity with different ratios of lysis buffer to sample (10:0, 9:1, 3:1, 1:1, 1:3, 1:9, 0:10). The real-time fluorescence curves of the 10-minutes single-step CRISPR reaction loaded with different volumes of lysed (n) rash fluid swab, (o) oral swab, (p) saliva and (q) urine sample (0, 1, 2, 5, 7 µL), and their (r) end-point fluorescence intensity. (s) meant that the viral lysis was easy-to-use and time-saving than the conventional nucleic acid extraction. The real-time fluorescence curves of the 10-minutes single-step CRISPR reaction using (t) rash fluid swab, (u) oral swab, (v) saliva and (w) urine samples processed by viral lysis and nucleic acid extraction, respectively. For (a-l) and (t–w), error bars represent mean ± s.d. for 3 technical replicates. For (n–q), error bands represent mean ± s.d. for 3 technical replicates. In (m, r), values represent the mean for 3 technical replicates.