Twistamp nfo kit

To test the suitability of the primers, RPA was performed in a 50 μL volume using a TwistAmp^® Basic kit (TwistDx, Cambridge, UK). The mixtures contained 10–50 ng of template DNA, 0.2 μM of each RPA primer, 1× rehydration buffer, and DNase-free water. A dry enzyme pellet was then added and thoroughly mixed. A total of 14 mM magnesium acetate was pipetted into the tube lids, followed by centrifugation to transfer the magnesium acetate into the tube in order to initiate the RPA mechanism at 40 °C for 20 min. The reactions were visualized by 2% agarose gel electrophoresis (AGE). For the purposes of probe screening, RPA was performed in a 50 μL volume using a TwistAmp^® nfo kit (TwistDx, Cambridge, UK). The P01 and P02 primers were adapted for the RPA assay by labeling the reverse P02 primer with a 5′ biotin residue and testing both primers for compatibility with internal RPA LF (lateral-flow) probes, which were designed according to the TwistDX guidelines. A solution containing 0.2 μM P2 primer, 0.1 μM P1 primer, and 0.12 μM LF probe in 1× rehydration buffer and DNase-free water was added to a dry pellet from the Twist Amp nfo kit (TwistDx, Cambridge, UK). The template DNA (1 ng) was added, and 14 mM of magnesium acetate was pipetted into the tube lids. Centrifugation was performed to initiate the RPA mechanism at 40 °C for 20 min. The reactions were then visualized by LFD.

The frmRPA-LF was performed with the TwistAmp nfo Kit (TwistDx, Cambridge, UK). The reaction mixture consisted of 2.1 μL of both frm-forward and frm-reverse primers (final concentration: 420 nM), 0.6 μL of the frm-probe (final concentration: 120 nM), 2.5 μL of magnesium acetate (MgAc; final concentration: 14 nM), 29.5 μL of the rehydration buffer, 10 μL of the purified Formosan Reeves’ muntjac genomic DNA (concentration of 27.7 ng/μL), and 3.2 μL of nuclease-free water. Finally, the nfo RPA freeze-dried pellet was added to the reaction, followed by a brief vortex and spin. To optimize the reaction temperature and time, the amplification reaction mixture was incubated in a Lifepro Gradient Thermal Cycler (Bioer Technology, Hang, China) at different reaction temperatures (25, 30, 35, 40, 45, and 50 °C) for 20 min. Different amplification durations (5, 10, 15, 20, 25, and 30 min) were tested at 37 °C, the temperature instructed by the instruction manual of the TwistAmp nfo Kit (TwistDx, Cambridge, UK).

A commercial TwistAmp™ nfo kit (TwistDX, Cambridge, UK) and lateral flow strip (USTAR, Hangzhou, China) were utilized in the M. bovis LFS RPA assay. The reaction volume was 50 μl including 29.5 μl of rehydration buffer, 2.1 μl of each RPA primer (uvrC-nfo-F and uvrC-nfo-R, 10 μmol/L), 0.6 μl of exo probe (uvrC-nfo-P, 10 μmol/L), and 2.5 μl of magnesium acetate (280 mmol/L). In addition, 1 μl of bacterial genomic DNA or standard DNA was used for the specific and sensitive analysis, while 2 μl of sample DNA was used for the clinical sample diagnosis. The LFS RPA reactions were incubated in an incubator block at 39°C for 5, 10, 15, and 20 min. The lateral flow strips were used to recognize the amplicons dual-labeled with FAM and biotin. The LFS RPA products were identified visually by using lateral flow strips according to the manufacturer’s instructions.