Bst 3.0 enzyme

LAMP reactions were prepared in single 0.2 mL PCR tubes in a laminar-flow hood. Each experiment was repeated independently three times. A 10× primer mix of LAMP primers consisting of 2 μM F3 and B3 primers, 16 μM FIP and BIP primers, and 4 μM FL and BL primers was prepared, aliquoted, and stored at −20 °C. Bst 3.0 reactions consisted of 2.5 μL 10× reaction buffer, 1.5 μL 100 mM MgSO₄, 3.5 μL 10 mM dNTPs, 2.5 μL 10× primer mix, 1 μL Bst 3.0 enzyme (NEB, #M0374S), and 2 μL 10 ng/µL DNA. Bst 2.0 Warm Start reactions consisted of 12.5 μL 2× Warm Start master mix (NEB #E1700S), 2.5 μL 10× primer mix, and 2 μL 10 ng/µL DNA. All reactions were assembled on ice. A no-template control was included to ensure amplification specificity. Reactions were incubated at either 65 °C for 30 min (Mth, Mco), 65 °C for 45 min (Mcu), or 58 °C for 45 min (Mbr) before the enzyme was heat inactivated at 80 °C for 5 min. Product detection was performed by carefully transferring 9 μL of the stopped reaction to a fresh 0.2 mL PCR tube and adding 1 μL of 1000× SYBR Green I dye (Thermo Fisher Scientific, Waltham, MA, USA). Visual inspection was performed with positive reactions turning yellow/green while negative reactions remained orange. DNA template amplification was additionally visualised by loading 5 μL of product on a 1.5% (w/v) agarose gel in 1× TAE following electrophoresis at 110 V for 45 min.

First, 10 ng/µL of genomic DNA was diluted by 10-fold serial dilution using sterile nuclease-free water to a concentration of 0.001 ng/µL. LAMP reactions with Mcu and Mco primers were performed in technical duplicates using 2.5 μL 10× reaction buffer, 1.5 μL 100 mM MgSO₄, 3.5 μL 10 mM dNTPs (source), 2.5 μL 10× primer mix (see LAMP assay section), 1 μL Bst 3.0 enzyme (NEB, #M0374S), and 1 μL (10 ng/μL–0.001 ng/μL) DNA. Bst 2.0 Warm Start reactions for Mth consisted of 12.5 μL 2× Warm Start master mix (NEB #E1700S), 2.5 μL 10× primer mix, and 2 μL 10 ng/µL DNA. Reactions were incubated at either 65 °C for 30 min (Mth, Mco) or 65 °C for 45 min (Mcu) before the enzyme was heat inactivated at 80 °C for 5 min. End-point products were visualised as described in the LAMP assay section.