Goldview

Polymerase chain reaction (PCR) was used to detect the highly conserved species-specific gene toxR and the virulence genes tdh and trh in all V. parahaemolyticus isolates (Law et al., 2017 (link)). Genomic DNA was extracted using a bacterial DNA extraction kit (Sangon, Shanghai, China) according to the manufacturer’s instructions. The primers used to detect toxR, tdh and trh are shown in Table 1. Each PCR amplification reaction was performed in a 25 μL mixture containing 250 ng of DNA as the template, 400 nM each primer, 200 mM each deoxynucleotide triphosphate (dNTP), 10 × PCR buffer, and 5 U of Ex-Taq DNA polymerase (Takara-Bio, Beijing, China). PCR amplification was initiated by incubating the reaction mixture at 94°C for 1 min, followed by 30 cycles at 98°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and a final extension at 72°C for 10 min (Jiang et al., 2019 (link)). PCR products (5 μL) were mixed with 1 μL of 6 × loading buffer dye and analyzed by electrophoresis on a 1.2% agarose gel containing GoldView (Sangon, Shanghai, China). V. parahaemolyticus isolates ATCC33847 (tdh⁺ and trh^–) and ATCC17802 (tdh^– and trh⁺) were used as positive control isolates, and distilled water was used as the negative control.

Total genomic DNA was extracted using a kit (Tiangen Biotech, China). All genomic DNA solutions were stored at -20℃. According to previous studies, 6 antibiotic resistance genes (ARGs), 13 mobile genetic elements (MGEs) and gene cassettes were selected and detected by polymerase chain reaction (PCR)^{19 (link)–22 (link)}. PCR assays were carried out in 25 μL volumes containing 2 μL template DNA, 12.5 μL 2 × Taq PCR Master Mix (Tsingke, China), 8.5 μL ddH₂O (Solarbio, China) and 1 μL each primer. The PCR products were separated by gel electrophoresis in a 1.5% agarose gel stained with GoldView™ (Sangon Biotech, China), visualized under ultraviolet light and photographed using a gel documentation system (BioRad, USA). The primers and amplification conditions used have been previously described in Table S1^{23 (link),24 (link)}.

Ticks were identified as D. nuttalli through morphological characteristics [17 (link)]. All D. nuttalli samples were individually extracted using a TIANamp Tissue and Blood Kit (TIANGEN, Beijing, China) [18 (link)]. For amplification of Rickettsia DNA, the gltA and ompA genes were amplified by polymerase chain reaction (PCR), following the protocols described by Bermúdez et al. [19 (link)]. Specific primers targeting the gltA and ompA genes of Rickettsia from D. nuttalli were synthesized by Shanghai Sangon. DNA was amplified using a system of 40 μl, each including Taq PCR Master Mix (Sangon, Shanghai, China), 2 μl of DNA from each sample, and 1 μl of each reverse and forward primer, and filled to volume with double-distilled water. The PCR primers, amplification sizes (base pairs), and annealing temperatures are listed in Additional file 1: Table S2. Double-distilled water was used as the negative control in each PCR reaction. Prior to sequencing, the quality of the PCR products was checked with 1.5% agarose gel electrophoresis stained with GoldView (Sangon, Shanghai, China). If the quality of the PCR product was suboptimal, it was purified using the Gel DNA Recovery Kit (TIANGEN, Beijing, China) and cloned using the pGEM-T Easy Vector System (Promega, Madison, WI, USA).