Superscrip 4 reverse transcriptase

Using the detection method developed above, RPA and CRISPR-Cas13a were detected in two steps. To further simplify the procedure while reducing the risk of contamination, we performed RT, RPA, and CRISPR-Cas13a detection in a single step. The reaction mix consisted of detection buffer, 29.5 μL of Primer Free Rehydration buffer, 100 U of RNase inhibitor, 200 U SuperScrip IV reverse transcriptase (Thermo Fisher, Waltham, MA, United States), 10 U of RNase H (NEB, Ipswich, MA, United States), 2.4 μL each of forward or reverse RPA primers, 45 nM of purified LwaCas13a protein, 22.5 nM of crRNA, 125 nM RNase reporter, 100 U of T7 RNA polymerase, 5 μL of RNA, and 8 mM of NTP mix, which were used to resuspend the freeze-dried reaction pellets. Thereafter, 5 μL of 280 mM MgOAc was added to complete the reaction. Fluorescence kinetics and lateral-flow-based readouts were collected as described above.