Bst 3

The LAMP reaction system contained 1 × Isothermal Amplification Buffer (New England Biolabs, Shanghai, China), 1.2 mM dNTP (Sangon Biotech, Guangzhou, China), 6 mM MgSO4 (New England Biolabs, Shanghai, China), 320 U mL⁻¹ Bst 2.0 DNA polymerase (New England Biolabs, Shanghai, China), 160 U mL⁻¹ Bst 3.0 (New England Biolabs, Shanghai, China). Primers used for the LAMP are shown in Table S2. A primer mix consisting of 1.6 μM of FIP and BIP, 0.2 μM of F3 and B3, and 0.4 μM of LF and LB was added to the reaction. RT-LAMP reactions were carried out in 0.2 mL PCR tubes. After adding the 1 μL sample, the final volume of the RT-LAMP reaction was 20 μL. In addition to the above reagents, the real-time LAMP reaction mixture contains 1 × LAMP Fluorescent Dye (New England Biolabs, Shanghai, China). The LAMP mixture was incubated at 65 °C for 50 min in a LongGene PCR System and the fluorescent signal was recorded every 1 min. The reaction mixture used in the system contains hydroxy naphthol blue (Aladdin, Shanghai, China).

The final 25 µL reactions, prepared with 1X Isothermal Amplification Buffer (I) (NEB Ipswich, MA, USA, B0537S) had 1.6 mM of both FIP and BIP primer pairs, 0.2 mM of influenza B or BPIFA1 F3 and B3 primer pairs, 0.25 µM of the NB-F3 and NB-B3 primer pair, 1 µM of both LF and LB primer pairs (LF or LB being modified with a fluorophore) and 2 mM of the corresponding LF or LB quencher primer, 6 mM MgSO4, 1.6 mM dNTPs, 1 µL (15 units) of WarmStart RTx (NEB, Ipswich, MA, M0380), and 2 µL (16 units) of Bst 3.0 (NEB, Ipswich, MA, USA, M0374). Reactions were incubated at 63 °C for 45 min.