Twistamp liquid basic kit

Two DNA amplification methods (PCR and RPA) were used in this study. PCR was conducted as a routine procedure. RPA was conducted on a dry bath incubator with the TwistAmp Liquid Basic Kit (TwistDx). Fifty microliters of the RPA reaction system contained 25 μL of buffer, 9.2 μL of dNTP (10 μM), 5 μL of E-mix, 2.5 μL of the core reaction mix, 2.5 μL of MgOAc (280 mM), 2.4 μL of forward and reverse RPA primer, respectively (10 μM), and 1 μL of DNA template. The amplification condition of RPA was 40 °C for 40 min.

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).

The one-tube RPA-Cas12a detection platform combines RPA amplification and Cas12a detection. The RPA amplification was performed at the bottom of the tube according to the TwistAmp Liquid Basic kit (TwistDx, Maidenhead, UK) with modifications. For a 25 $\mathsf{\mu }$ L of RPA reaction contains a premix of 10 $\mathsf{\mu }$ L of 2× Reaction Buffer, 1 $\mathsf{\mu }$ L of dNTPs, 2 $\mathsf{\mu }$ L of 10× E-mix and 1 $\mathsf{\mu }$ L each of RPA primer F and R; 1 $\mathsf{\mu }$ L of 20× Core Reaction mix, 2 $\mathsf{\mu }$ L of template DNA; 2 $\mathsf{\mu }$ L of MgOAc was added at the end. In addition, the CRISPR/Cas12a system was put at the tube lid, which included 1 $\mathsf{\mu }$ L of LbaCas12a (1 $\mathsf{\mu }$ M), 1 $\mathsf{\mu }$ L of CrRNA (2 $\mathsf{\mu }$ M) and 0.25 $\mathsf{\mu }$ L RNA inhibitor, 1 $\mathsf{\mu }$ L ssDNA FQ Reporter and 2 $\mathsf{\mu }$ L of 10× NEBuffer 2.1. The tubes should be covered gently, placed on a thermostat and incubated at 42 ${}^{\circ }$ C for 10 min to perform the PRA amplification reaction. At that time, the Cas12a system was briefly spun down and mixed with the RPA amplicons, and they were immediately placed on the LightCycler 96 system and incubated at 42 ${}^{\circ }$ C for real-time fluorescence acquisition. To monitor the real-time progress of the reactions, the fluorescence intensity was recorded every 1 min.