Conventional PCR

The primers were supplied from Biobasic (Canada) and are listed in Table-1.

Primers sequences, target gene, amplicon sizes, and cycling conditions for conventional PCR.

PCR=Polymerase chain reaction

The primers were utilized in a 25-μL reaction containing 12.5 μL of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 μL of each primer at a 20 pmol concentration, 4.5 μL of water, and 6 μL of DNA template. The reaction was performed on an Applied Biosystem 2720 thermal cycler.

The PCR products were separated by electrophoresis on a 1% agarose gel (Applichem, Germany, GmbH) in 1× TBE buffer at room temperature using a current of 5 V/cm. For gel analysis, 15 μL of the products were loaded in each gel slot. A 100-bp DNA ladder (Fermentas, Thermo, Germany) was used to determine the fragment sizes. The gel was photographed using a gel documentation system (Alpha Innotech, Biometra), and the data were analyzed using computer software.

TaqMan RT-PCR was done on serum samples using primers and probes [15] targeting the bcsp31 gene (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"M20404","term_id":"144104","term_text":"M20404"}}M20404). Primers’ sequences, target gene, and TaqMan probe are listed in Table-2.

Primers sequences, target gene, and cycling conditions for TaqMan RT-PCR.

RT-PCR=Real-time polymerase chain reaction

TaqMan RT-PCR

The primers were used in a 25-μL reaction containing 12.5 μL of the HERA q-PCR PCR Master Mix (Willowfort, UK), 0.2 μL of each primer at a 20 pmol concentration, 0.1 μL of the probe, 7.0 μL of DNase free water, and 5 μL of DNA template. The reaction was performed in a Thermo Scientific Piko RT-PCR machine (Thermo Fisher Scientific, US). The DNA of Brucella-positive and -negative controls were included in each run to indicate any amplicon contamination or amplification failure.