Because temperature control can be a notable factor in point-of-care assays, we next analyzed the effect of lysis temperature on CRISPR-FDS signal. No signal was detected when lysis reactions were performed at room temperature (~22°C), similar CRISPR-FDS signal was detected at 37° and 65°C, the manufacturer-recommended lysis temperature, with the most signal detected at 95°C (Fig. 1C). We next examined the effect of lysis time on CRISPR-FDS signal when using a 1:1 saliva-to-buffer ratio at 65°C and found that signal increased with incubation time out to the final 15-min time point (Fig. 1D). Given that there was no difference between 65° and 37°C lysis, we elected to use 37°C for the lysis temperature, with the assumption that this minimum temperature would be the simplest to achieve, maintain, and measure in most point-of-care settings, although temperature deviations above this minimum would not negatively influence the lysis reaction. We also chose to use a 5-min lysis time to minimize overall assay performance time.
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https://advances.sciencemag.org/content/7/2/eabe3703