Escherichia coli exonuclease 3

RPA reactions were performed using the TwistAmp Liquid Basic kit (TwistDx, Cat #TALQBAS01), according to the manufacturer's recommendations, with final concentrations of 0.6 μM of each primer (Thermo Fisher UK, Cat #10336022), 0.12 μM probe (LGC Biosearch Technologies, Risskov, Denmark, Cat #RPA-BF-2), 2 U/μL Escherichia coli Exonuclease III (NEB, Hitchin, UK, Cat #M0206L), 14 mM MgAc, and 1 μL of DNA template, in a total of 5 μL/reaction for limit of detection and specificity experiments. For extracted clinical samples and FTA card amplifications, 25 μL reactions were used, since their use yielded more consistent results over 5 μL reactions. Amplification was visualized with a Bio-Rad CFX Connect Real-Time PCR detection system (Bio-Rad, Cressier, Switzerland, Cat #1855201), running for 20 min at 37°C, with a pause at 4 min where the reactions were manually agitated (by inversion 10-times), a step which is favorable for reaction kinetics (26 (link)). Fluorescence data acquisition was taken once every 30 s (i.e., once per cycle) for a total of 40 cycles (20 min).