Dntps

In our study, chemicals and reagents used were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). In addition, Oligo (dT), dNTPs, and M-MLV reverse transcriptase were collected from Tiangen (Beijing, China).

DPPH and Trypan blue dye, DAPI, Methanol (Sigma, USA); BHT and DMSO (Merck, Germany); M-MLV reverse transcriptase, 1^st strand buffer, dNTPs, and primer of oligo (dT), p53, Bax, caspase-3, Bcl-2 and β-actin (Tiangen Biotech, Beijing, China).

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.

Genomic DNA was extracted from endophytic bacterial cultures through enzymatic hydrolysis (Saito and Miura, 1963 (link)). The complete 16S rRNA gene (1.4–1.5 kb) was amplified via PCR, using universal bacterial primers 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1541R (5′-AAG GAG GTG ATC CAG CCG CA-3′). The amplification was carried out on a DNA engine gradient thermal cycler (BIO-RAD, USA). The 50 μL PCR reactions contained 4 μL of 2.5 U/μLTaq DNA polymerase (Tiangen, Beijing), 5 μL of 10× buffer (Tiangen), 1 μL of 20 mM dNTPs (Tiangen), 37 μL of SDW, 1 μL of 50 μM each primer, and 1 μL of the template.
The PCR products were purified and sequenced by SolGent (SolGent, Daejeon, Korea). The PCR conditions were initial denaturation at 94°C for 4 min, 30 amplification cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min, and final elongation at 72°C for 10 min. Sequences were compared with published data in the GenBank databases (http://www.ncbi.nlm.nih.gov/genbank/). CLUSTAL-W in MEGA 6.06 software was used to align closely related sequences (Tamura et al., 2013 (link)). The neighbour–joining (NJ) method was employed to construct a phylogenetic tree for 16S with MEGA 6 after sequence alignment with Clustal W (version 7.222) (Katoh and Standley, 2013 (link)). Each node in the phylogenetic trees was statistically supported using 1000 bootstrap replications.