Bst3.0 polymerase

LAMP was carried out for each strain in a total 25 µl reaction mixture containing 1.6 µM each FIP and BIP, 0.2 µM each F3 and B3, 0.6 µM each FL and BL, 200 µM each dNTP (roboklon, EURx, Cat.No. E0503-01), 0.4 M betaine, 1 × Isothermal buffer (New England Biolabs, B0537), 6 mM MgSO₄ (New England Biolabs, B1003) and 0.32 units/µl Bst3.0 polymerase (New England Biolabs, M0374L). Depending on the experiment the mixture was incubated at 65 °C for an amplification time of 20–30 min, then heated at 85 °C for 3 min to terminate the reaction.
Aminoallyl-dUTP-5/6-TAMRA (Jena Bioscience, NU-803-TAM) were used for labeling of LAMP products with final concentrations ranging from 10 µM to 80 µM (0.5–4%) per reaction. The amount of nuclease free water was adjusted to fit 25 µl reaction volume. Each LAMP reaction contained 1µl of genomic DNA with varied concentrations according to the experiment. For the analysis of LAMP reaction in real-time (rtLAMP) (Analytik Jena, qTOWER³G touch thermocycler, Farbmodul 1), 1µl of 50 × SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific; S33102) was added per reaction.
As part of the experiments, non-templated controls (NTC) were carried out. These controls contained all components of a LAMP reaction except for the inclusion of the genomic DNA template.