Uv transilluminator

Specific primers were used to amplify sequences of 6 different virulence genes. Primer sequences and predicted sizes of the PCR products are shown in (Table 1). The amplification reactions were carried out using Biometra, UNO II thermal cycler (Goettingen, Germany) under the following conditions: initial denaturation at 95°C for 5 min, followed by 35 cycles of: 30 s at 94°C for, 30s at 63°C, then 30 s at 72°C, with a final extension step at 72°C for 5 min. PCR was performed in a 25 ml reaction mixture containing1 ul of template DNA (*100 ng/ml), 12.5 ml of PCR mastermix (Maxima Hot Start Green PCR Master Mix, USA), and 1 ul (10 pmol) of each primer and 9.5 ml of nuclease free water. PCR products were resolved on 2% agarose gel and visualized under a UV transilluminator (Biometra).

For the molecular assay, the DNA template was equipped with the boiling method as described by Queipo-Ortuño et al. [24 (link)]. The 16S rRNA gene-based PCR was performed for the confirmation of the genus Salmonella. Primers were used for the amplification of the 16S rRNA gene according to the procedure described by Lin and Tsen [25 (link)] and shown in Table-1. The reaction mixture (20 µl) was prepared by mixing 10 µl master mixtures (Promega, USA), 1 µl forward primer (10 pmol), 1 µl reverse primer (10 pmol) (BioServe Biotechnologies Ltd., USA), 3 µl DNA template, and 5 µl deionized water. The PCR reactions were carried out using a thermocycler (Astec, Japan) with the following program: Initial denaturation with one cycle for 5 min at 94°C, 30 cycles each consist of denaturation with 30 s at 94°C, annealing with 30 s at 50°C, extension with 30 s at 72°C, and a final extension step of 5 min at 72°C. PCR products were analyzed by 2% agarose (Invitrogen, USA) gel electrophoresis and the bands were visualized with ultraviolet (UV) light after staining with ethidium bromide (0.5 µg/ml) for 10 min in a dark place. Bands were visualized and images were captured on a UV transilluminator (Biometra, Germany).

This step involved the multiplex polymerase chain reaction (mPCR). Three primers targeting the Campylobacter 23S rRNA gene, the hip O gene for C. jejuni, and the glyA gene for C. coli were used to amplify 650, 323, and 126 bp (Supplementary Figure S1), respectively, as described previously (19 (link)). PCR was carried out in a volume of 50 μl with 25 μl of the master mix, 10 μl of the DNA template (50 ng), 9 μl of grade water, and 1 μl of each primer (20 pmol). The PCR conditions were as described previously (19 (link)), as follows: there was an initial denaturation step of 94°C for 6 min, followed by 35 cycles, each consisting of 30 s at 95°C, 30 s at 59°C, 30 s at 72°C, and a final extension step at 72°C for 7 min. The electrophoresis of PCR products was performed in 1.5% agarose at 80 V in Tris base–boric acid–EDTA buffer for 120 min. The UV trans-illuminator (Biometra) was used for the visualization of amplicons. Then, they were photographed with the Gel Documentation System using BioDocAnalyze software. A negative control and a positive control were also included.

The amplified products were then separated by agarose gel electrophoresis. PCR-products were run on 1% agarose gels (HiMedia, Mumbai, India) containing Ethidium Bromide stain (EtBr) (HiMedia, Mumbai, India) with 1x TAE buffer (40 mM Tris- HCl, 20 mM Naacetate, 1mM EDTA, pH 8.4) and the bands were visualized under an UV transilluminator (Biometra, Germany). Images were captured with digital imaging system (Bio-Rad).

DNA template of each isolate of Campylobacter spp. was prepared by boiling method described by Hoshino et al. (1998) [26 (link)]. In brief, a pure bacterial colony grown on blood based agar was mixed gently in 250 μL distilled water and subjected to boiling, followed by immediate cooling on ice, for 10 min each. The tubes were then centrifuged at 10,000X g for 10 min and the supernatant was collected as DNA template for polymerase chain reaction (PCR). Initially, PCR screening targeting 16S rRNA gene was performed according to Samosornsuk et al. (2007) [27 (link)] to confirm whether the strains belonged to the genus Campylobacter. Afterwards, cdtC gene based multiplex PCR was done for species-specific (C. jejuni, C. coli and C. fetus) identification following methods described by Asakura et al. (2008) [28 (link)]. DNA templates of C. jejuni ATCC33560, C. coli ATCC33559 and C. fetus ATCC27374 strains were used as positive controls, and that of Escherichia coli ATCC 25922 was used as a negative control. Details of all primers and corresponding PCR amplicon sizes are shown in Additional file 1. PCR products were subjected to gel electrophoresis (1.5% agarose, Invitrogen, USA) and after staining with ethidium bromide (0.5 μg ml^− 1) and destaining with distilled water, each for 10 min, gel images were captured using a UV transilluminator (Biometra, Germany).