Optimizing LAMP Assay for L. pneumophila

Optimization of the LAMP assay was performed in a 25 µL reaction mixture containing 2.5 µL 10× Bst buffer (New England Biolabs, Ipswich, MA, USA), 0.2 pmol each of F3 and B3, 0.8 pmol each of FIP and BIP, different concentrations of dNTPs (0.4, 0.6, 0.8, 1, and 1.2 mM), different concentrations of MgSO₄ (3, 4, 5 and 6 mM), 8 U Bst DNA polymerase (New England Biolabs), and different concentrations of DNA template. The reaction mixture was incubated at 60°C–65°C for different times and heated to 95°C for 5 minutes to stop Bst DNA-polymerase activity in the reaction. L. pneumophila serogroup 1 (ATCC 33152) was used as the positive control. Finally, LAMP-reaction products were electrophoresed on 2% agarose gel for detection of best LAMP product. After optimization of the LAMP assay, the amplified products were detected by adding 1 µL 1:10 SYBR green I (Thermo Fisher Scientific) into a reaction microtube. The reaction was considered positive if its color turned from orange to green under ultraviolet light.16 (link)