In total, 19 known bacterial strains, including a V. parahaemolyticus reference strain (ATCC 17802) and seven Salmonella sp. strains, were analyzed in this study (Table 1 ). All strains were stored in 10% (w/v) glycerol broth at −70 °C. Salmonella typhimurium CICC 21483 and V. parahaemolyticus ATCC 17802 strains were used for primer optimization and sensitivity testing. Vibrio strains were cultured in trypticase soy broth supplemented with 2% NaCl at 35 °C overnight. Non Vibrio strains were grown in Luria-Bertani broth at 37 °C.
Genomic DNA extraction was conducted using the boiling method. Briefly, 1 ml of overnight bacterial culture was centrifuged at 5,000 × g for 10 min and the pellet was suspended in 200 μl of TE buffer [10 mM Tris, 0.1 mM EDTA (pH 8.0)]. The bacterial suspension was boiled for 10 min and centrifuged at 12,000 × g for 5 min at 4 °C. The supernatant was used as the DNA template for multiplex LAMP and multiplex PCR assays. The genomic DNA of S. typhimurium CICC 21483 and V. parahaemolyticus ATCC 17802 was extracted with the Genomic DNA Isolation Kit (Sangon Biotechnology, Shanghai, China) following the manufacturer’s instructions.
Genomic DNA extraction was conducted using the boiling method. Briefly, 1 ml of overnight bacterial culture was centrifuged at 5,000 × g for 10 min and the pellet was suspended in 200 μl of TE buffer [10 mM Tris, 0.1 mM EDTA (pH 8.0)]. The bacterial suspension was boiled for 10 min and centrifuged at 12,000 × g for 5 min at 4 °C. The supernatant was used as the DNA template for multiplex LAMP and multiplex PCR assays. The genomic DNA of S. typhimurium CICC 21483 and V. parahaemolyticus ATCC 17802 was extracted with the Genomic DNA Isolation Kit (Sangon Biotechnology, Shanghai, China) following the manufacturer’s instructions.