FMDV RNA Rapid Isothermal Detection

LFS RT-RPA reactions were performed in a 50 μL volume containing 29.5 μL rehydration buffer and 2.5 μL magnesium acetate (280 mM) from the TwistAmp™ nfo kit (TwistDX, Cambridge, UK). Other components included 420 nM each RPA primer (FMDV-LFS-F and FMDV-LFS-R), 120 nM LF probe (FMDV-LFS-P), 200 U MMLV reverse transcriptase (Takara, Dalian, China), 40 U Recombinant RNase Inhibitor (Takara, Dalian, China) and 1 μL of viral RNA or 5 μL of sample RNA. Except for the viral template and magnesium acetate, the other reagents were prepared in a master mix and distributed into a 0.2 mL freeze-dried reaction tube containing a dried enzyme pellet. One μL of viral RNA and 2.5 μL of magnesium acetate were pipetted into the tubes. The RPA was performed in the technician’s closed fist at room temperature for 5, 10, 15 and 20 min as described previously [26 (link), 27 (link)]. The RPA products, which were dual labelled with FAM and Biotin, were detected using LFS as described previously [26 (link), 27 (link)]. A testing sample was considered positive when both the test line and the control line were visible, negative when only the control line was visible, and invalid when the control line was invisible.