Safe stain

Nested PCR was conducted using specific primers for the Nc-5 gene. The first round of PCR was conducted using a pair of N. caninum–specific primers, Np21plus (5’-CCCAGTGCGTCCAATCCTGTAAC-3’) and Np6plus (5’-CTCGCCAGTCCAACCTACGTCTTCT-3’) (Muller et al. 1996 ). Nested PCR was performed with the primers Np6 (5’-CAGTCAACCTACGTCTTCT-3’) and Np7 (5’-GGGTGAACCGAGGGAGTTG-3’) (Hughes et al. 2006 (link)). Each amplification was performed in 20-µL reaction mixtures containing 10 µL of 2x master mixes (DFS Master Mix, BIORON GmbH, Ludwigshafen, Germany), each of the respective primers (10 pmol for the first round reaction and 25 pmol for nested PCR), 7 µL of distilled water, and 1 µL of template DNA. One microlitre of the first round product was used as the template for nested PCR. For each reaction, a negative control (double distilled water) and a positive control (DNA extracted from the Nc-5 strain of N. caninum) were included. Amplification was performed with initial denaturation for 5 minutes at 94 °C, followed by 40 cycles at 94 °C for 40 seconds (denaturation), annealing at 62 °C in the first round, and 56 °C in nested PCR for 40 seconds, extension at 72 °C for 40 seconds, and final extension at 72 °C for 10 minutes. PCR products were electrophoresed on a 1.5% agarose gel stained with safe stain (Sinaclon, Tehran, Iran) and visualised under ultraviolet trans-illumination.

We used the specific primers (Metabion, Germany) including veb-forward-5′-CGACTTCCATTTCCCGATGC-3′ and veb-revers-5′-GGACTCTGCAACAAATACGC-3′ for detection of bla_VEB gene with a product size of 642 bp [23] (link), per-forward-5′-ATGAATGTCATTATAAAAGC-3′ and per-revers-5′-TTAATTTGGGCTTAGGG-3′ for detection of bla_PER gene with a product size of 933 bp [24] (link), and ges-forward-5′-GTTTTGCAATGTGCTCAACG-3′ and ges-revers-5′-TGCCATAGCAATAGGCGTAG-3′ for detection of bla_GES gene with a product size of 387 bp [25] (link) by PCR test. The PCR reaction was carried out in a final volume of 15 µL containing 7.5 µL of PCR Master Mix (Ampliqon, Denmark), 5 pmol of each primer, 5.5 µL of distilled water, and 300 ng of DNA. The initial denaturation step was done at 94 °C for 5 min. Then, 34 cycles of the amplification were performed as follows: Denaturation at 94 °C for 45 sec, annealing step for 20 sec at 63 °C for bla_VEB, 55 °C for bla_GES, and 52 °C for bla_PER, and extension at 72 °C for 25 sec. A final extension step was used at 72 °C for 10 min. Then, the PCR product was electrophoresed on 1% agarose gel (Wizbiosolutiotions) with 1.5 µL of Safe Stain (SinaClon, Iran).

The buffy coat samples were used for DNA extraction using the phenol–chloroform–isoamyl alcohol method, as described in a previous study [16 (link)]. PCR was performed using the repetitive element (RE) gene amplifying a region of 529 base pairs (bp) fragments. The primers are highly sensitive and specific for T. gondii due to 200–300 replications in the T. gondii genome [17 (link)]. The PCR primers and cycling conditions were described in previous reports [18 (link), 19 (link)]. For each reaction, a positive control (DNA extracted from the RH strain of T. gondii) and a negative control (double-distilled water) were included. PCR products were electrophoresed in 2% agarose gel, stained with safe stain (Sinaclon, Iran), and visualized under a UV transilluminator.