2.4. Virulotyping by Polymerase Chain Reaction (PCR)

Virulence gene profiling (i.e., virulotyping) in Salmonella isolates (n = 211) was done by end-point Polymerase Chain Reaction (PCR) targeting 13 selected virulence genes (avrA, bcfC, gipA, invA, mgtC, pefA, sefA, siiD, sodC1, sopB, sopE1, spvC, and ssaQ). We also tested an integron-associated integrase class 1 (intI1) gene marker in these isolates. Five virulence gene targets (avrA, ssaQ, mgtC, siiD, and sopB) were located on the Salmonella pathogenicity islands (SPIs) 1–5, three targets (gipA, sodC1, and sopE1) were located on prophages, one (spvC) was located on the S. Typhimurium virulence plasmid, and one (bcfC) was located on a fimbrial cluster [28]. Another target virulence gene, invA, is an invasion gene of the genus Salmonella; sefA is a fimbrial antigen of S. Enteritidis, and pefA is a plasmid-encoded fimbria of S. Typhimurium [29]. The integron-associated class 1 integrase gene (IntI1) is often located on transposons containing two to eight gene cassettes encoding resistance to a broad spectrum of antibiotics [15].

Multiplex PCR was used to amplify the DNA of each Salmonella isolate targeting the 13 virulence genes and integron-associated class 1 integrase gene (IntI1). Primers and DNA sequences of the genes can be found in Supplementary Table S1. Nine PCRs were completed based on the base-pair size of the primer and annealing temperature associated with the virulence gene. The virulence genes avrA, mgtC, and sopB were included in the first multiplex PCR. In the PCR reaction, 5 μL of genomic DNA template was added to 25 μL of 2× PCR Master Mix (Promega, Madison, WI, USA), 2 μL of nuclease-free water, and 1 μL of each primer (10 μM) [28]. The cycling conditions were as follows: 95 °C for 1 min, followed by 30 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, ending with a final extension step at 72 °C for 4 min and followed by a hold at 4 °C. Separate multiplex PCRs were completed for combinations of (gipA and sopE1), (sodC1, spvC, and bcfC), and (ssaQ and siiD) using the same cycling conditions. Another multiplex PCR was completed for invA and sefA. The reaction had a final volume of 25 μL containing PCR reaction buffer (50 mM KCl, 10 mM Tris-HCl, 2.5 mM MgCl₂, pH 8.3), 200 μM dNTPs, 0.2 μM invA primers, 0.2 μM sefA primers, and 0.5 μM pefA primers, 2.5 U of Taq DNA polymerase (MBI Fermentas), and 0.2 μL of DNA template [29]. The cycling conditions consisted of denaturation for 30 s at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C for 35 cycles, followed by a final extension for 7 min at 72 °C. The target virulence gene pefA followed the same conditions in a separate PCR. The final PCR targeted the integron-associated integrase class 1 with the following cycling conditions: denaturation at 94 °C for 3 min; 94 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s, 35 cycles; 72 °C for 5 min [30]. There were two negative controls for each PCR with a template consisting of nuclease-free water. Gel electrophoresis of amplicons was completed using a 2% agarose gel containing 0.5 μg/mL of ethidium bromide to check the integrity of the DNA. A UV transilluminator was used to visualize the amplified DNA fragments for analysis.