Fig. 2: Optimization of the single-step SHERLOCK reaction.
From: Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2
a Background-subtracted fluorescence of Cas13-based detection with synthetic RNA, reverse transcriptase, and RPA primers (but no RPA enzymes) after 3 h. b Single-step SHERLOCK normalized fluorescence using various buffering conditions after 3 h. c Background-subtracted fluorescence of single-step SHERLOCK with synthetic RNA and variable RPA forward and reverse primer concentrations after 3 h. d Single-step SHERLOCK normalized fluorescence over time using two different fluorescent reporters (left) and two different reverse transcriptases (right). e Background-subtracted fluorescence of the original single-step and optimized single-step SHERLOCK with synthetic RNA after 1 h. Data from the 3-h timepoint from this experiment are shown in Fig. 1d. f Colorimetric detection of synthetic RNA input using optimized single-step SHERLOCK after 3 h. Max maximum test band intensity, 5698.4 a.u., Min minimum test band intensity, 104.4 a.u. g Optimized single-step SHERLOCK background-subtracted fluorescence using RNA extracted from patient samples after 1 h. h Concordance between SHERLOCK and RT-qPCR for 7 patient samples and 4 controls. For c, e, see “Methods” for details about normalized fluorescence calculations. For b, d, f, g, NTC non-template control. For a, c, center = mean for 2 technical replicates. For d–f, center = mean and error bars = s.d. for 3 technical replicates. For b, d, RNA input at 104 cp/μL. For a–e, g, source data are provided as a Source data file.
