LAMP Reaction

Loop-mediated isothermal amplification reaction was performed in a 25 μl reaction mixture. The mixture contained 1 × ThermoPol Buffer (contained 2 mmol/L MgSO4), 6 mmol/L MgSO4 (total 8 mmol/L), 1.4 mmol/L of each dNTP, 1.6 μmol/L of each inner primers (FIP and BIP), 0.2μmol/L of each outer primers (F3 and B3), 8 units of Bst DNA Polymerase (Large Fragment).

When test the LAMP primer designed for S. aureus, the mixture contained 20 ng of template DNA of each 29 bacterial strains. When test the LAMP primer’s specificity designed for Vibrio vulnificus and Vibrio cholerae, the mixture also contained 20 ng of template DNA of each 29 bacterial strains and when test its commonality, the amount of template DNA was 50 ng, 5 ng, 500 pg, 50 pg, 5 pg, 500 fg, 50 fg, 5 fg and 0.5 fg, respectively. In all NTC (no template control) reaction, template DNA was replaced by sterilized water.

The LAMP reaction was carried out at 62°C for 60 min using a Veriti^TM Dx Thermal Cycler (Thermo Fisher, United States), then inactivated Bst DNA Polymerase at 80°C for 10 min. After the reaction, 1 μl 1000× SYBR Green I was added into the solution to confirm whether the reaction occurred. In a positive reaction, the color of the solution was green and in a negative reaction, the color was orange.