Sybr green 1

The DNA oligonucleotides were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and purified by high-performance liquid chromatography. Taq DNA ligase (40 U μL⁻¹), 10 × Taq DNA ligase reaction buffer (200 mM Tris-HCl, 250 mM KAc, 100 mM Mg(Ac)₂, 100 mM DTT, 10 mM NAD, 1% Triton X-100, pH 7.6), Bst 2.0 DNA polymerase (8 U μL⁻¹) and 10 × ThermoPol reaction buffer (200 mM Tris-HCl, 100 mM (NH₄)₂SO₄, 500 mM KCl, 20 mM MgSO₄, 1% Tween-20, pH 8.8) were all purchased from New England Biolabs (Beijing, China). SYBR Green I was obtained from Generay Biotech. Co., Ltd. (Shanghai, China). DNA marker, 6 × loading buffer and dNTPs were purchased from Takara (Dalian, China). TIANamp Genomic DNA Kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The nuclease-free water was purchased from Thermo Fisher Scientific Inc (Vilnius, Lithuania) and used in all ligation reaction and ligation-initiated LAMP assays. All the reagents were of analytical grade and used without further purification. All solutions were prepared with ultrapure water from Millipore Milli-Q water purification system (Millipore, USA). The detailed oligonucleotide sequences were listed in Table S1 (Supporting Information).

Bst DNA polymerase (large fragment) and dNTPs were obtained from Tiangen Biotech (China). SYBR Green I and the EasyPure Genomic DNA Kit (GK1071) were procured from Generay Biotech Co., Ltd. (China). Primers used in the study were custom synthesized from Generay Biotech. Biowest agarose was obtained from Thermo Fisher Scientific (USA). A universal rapid dipstick kit made by Milenia Biotec GmbH (Milenia GenLine HybriDetect, Germany) was used to test the LAMP amplicons by LFD.
V. parahaemolyticus ATCC 17802 was employed to develop the LAMP-LFD assay. Pure culture stocks of V. parahaemolyticus were retrieved from the glycerol stock (-80°C) and cultured in thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates at 37°C for 18-24 h. It was enriched by inoculation into 5 ml of alkaline peptone water (APW, Land Bridge Technology, China) containing 3% (w/v) NaCl. Seven non-Vibrio parahaemolyticus reference strains (Table 1) were used as control in this study. The strains of Vibrio vulnificus and Staphylococcus aureus were enriched in APW medium containing 3% (w/v) NaCl at 37°C for 18-24 h. The strains of Pseudomonas aeruginosa, Proteus mirabilis, Candida albicans, Streptococcus pyogenes and Salmonella enteritidis were enriched in APW medium at 37°C for 18-24 h.