2.4. DNA Extraction and Screening of Antimicrobial Resistance Genes

Genomic DNA of bacterial isolates was extracted through boiling methods. Briefly, a smooth, single colony was inoculated in 5 mL of nutrient broth and incubated at 37 °C for 12 h, and then 200 µL of overnight culture was mixed with 800 µL of distilled water and boiled for 10 min in a heat block. After boiling, the tubes were immediately placed on ice for 5 min followed by centrifugation at 14,000 rpm for 5 min. The supernatant, which contained bacterial DNA, was transferred to a new tube and stored at −20 °C [21]. All isolates were tested more than once for the presence of genes that were resistant to integrons (class1 and 2), ß-lactamases (bla_CTX-M and bla_TEM), and florfenicol (floR). Primer sequences, target genes and polymerase chain reaction (PCR) products are summarised in Table 1.

Primer names, target genes, oligonucleotide sequences, and the product sizes used in PCR and DNA sequencing.

Several PCR protocols were used to detect the target genes of the isolates (Table 2).The PCR products were loaded onto 1.0% agarose gel (Sigma-Aldrich Co., St. Louis, MO, USA) that was stained with 0.5 µg/mL ethidium bromide (Sigma-Aldrich Co., St. Louis, MO, USA). The amplified DNAs were electrophoresed at 100 V for 60 min on a mini, horizontal electrophoresis unit (Bio-Rad, Hercules, CA, USA). The gel was then visualised and photographed under an ultraviolet transilluminator.

PCR amplification and DNA sequencing.