Twistamp liquid exo kit

For real-time RPA, the TwistAmp Liquid Exo kit (TwistDx, Maidenhead, UK) was used according to the manufacturer’s instructions. The optimized reaction mix consisted of 1× reaction buffer, 0.45 mM of each dNTP, 1× Probe E-mix, 600 mM of each RPA primer, 200 nM of RPA probe, 1× Core Reaction Mix, 1× Exo, and 19 nM of MgOAc in a total volume of 50 µl with 1 µl of crude extract. Reaction incubation and fluorescence signal reading were performed using fluorimeters AmplifyRP^® XRT (Agdia, Île-de-France, France) or Genie^® II (OptiGene, Horsham, UK), consisting of 45°C for 35 min. All mixtures were shaken 5 min after the start of the reaction.

After hybridization of the probe with the template, the real-time detection was based on the cleavage of tetrahydrofuran (THF) between the fluorophore and the quencher thanks to E. coli exonuclease III. The real-time RPA assay was performed in a 50 µL volume using the TwistAmp^® Liquid Exo kit (TwistDx, Cambridge, UK). The reaction mixture included 25 µL of 2× reaction buffer, 2 µL of forward Meas_NS1 primer (10 µM), 2 µL of reverse Meas-NR1 primer (10 µM), 0.6 µL of Meas_Probe (10 µM), 1 µL of ROX 50× (Thermo Fischer Scientific, Illkirch, France), 3.4 µL of dNTPs (25 mM, Thermo Fischer Scientific), 5 µL of probe E mix (TwistDx), 1 µL of SuperScript II (200 U/µL Thermo Fischer Scientific), 2.5 µL of core reaction (TwistDx), and 1 µL of Exonuclease III (TwistDx). Extracted RNA (4 µL) was added to each tube. The addition of 2.5 µL of magnesium acetate (280 mM) initiated the RPA reaction. After 4 min of incubation at 42 °C in a Biometra TAdvanced (Analytik Jena GmbH, Jena, Germany), the reaction was carried out in a Step One Plus Applied Biosystem device (Thermo Fischer Scientific) at a temperature of 42 °C. Real-time detection was performed by quantifying the fluorescent signal ratio (FAM [6-carboxyfluorescein]/ROX [carboxyrhodamine]) every minute. The ROX passive reference fluorochrome was added to the reaction to normalize the well-to-well signal differences.

The real-time RPA assay was performed in a 50 μL volume using the TwistAmp^® Liquid Exo kit (TwistDx, Cambridge, UK). The reaction mixture included 25 μL of 2× reaction buffer, 2.1 μL forward primer (10 µM), 2.1 μL reverse primer (10 µM), 0.6 μL probe (10 µM), 1 µL ROX 50× (ThermoFischer Scientific, Illkirch, France), 2.6 μL dH₂O, 3.6 µL dNTPs (25 mm, ThermoFischer Scientific), 5 µL probe E mix (TwistDx), 2.5 µL of Core reaction (TwistDx), and 1 µL Exonuclease 3 (TwistDx). Extracted DNA (2 µL) was added to each tube. The addition of 2.5 μL magnesium acetate (280 mm) initiated the RPA reaction. After an incubation for 4 min at 39 °C in a thermostat C (Eppendorf, Montesson, France), the reaction was performed in a Step One Plus Applied Biosystem device (ThermoFischer Scientific) at a temperature of 39 °C.
Real-time detection was performed by quantifying the fluorescent signal ratio (FAM [6-carboxyfluorescein]/ROX [carboxyrhodamine]) every 30 s. The ROX passive reference fluorochrome was added to the reaction to weight the well-to-well signals.