Dna safe stain

DNA extraction was performed using a DNA extraction kit (Stool DNA Isolation mini Kit, Yekta Tajhiz Azma Co. Iran) based on the protocol of the manufacturer. The LAMP technique was performed targeting the highly conserved ITS2 gene of Toxocara spp. with a set of 4 primers (Table 1; Macuhova et al., 2010). LAMP reaction was carried out in a final volume of 25 μl (Fallahi et al., 2014). Since the loop primers were not designed for the ITS2 gene of both T. canis and T. cati, in the LAMP reaction, double‐distilled water replaced loop primers. The reaction tubes were placed in a water bath at 65°C for 1 hr. To the visual assessment of the LAMP amplicons, 3 µl of SYBR Green I (Invitrogen, Thermo Fisher Scientific Invitrogen lot, Carlsbad, California, United States) diluted in dimethyl sulfoxide (DMSO; Sigma‐Aldrich, St. Louis, Missouri, United States), was added to each tube and observed under daylight and UV light. Furthermore, the gel electrophoresis was performed on the LAMP products in a 1.5% agarose gel stained with DNA safe stain (1 µg/ml; Sinaclon Co., Tehran, Iran) and visualized under UV light. The assay repeatability was evaluated by performing LAMP reactions with duplicates for all stool samples. Genomic DNA from Toxocara spp. standard strain and doubled distilled water were included in each LAMP reaction as positive and negative controls, respectively.

With the usage of the DNA extraction kit (Roche, Germany, Lot. No. 10362400), genomic DNA was extracted from single bacterial colonies, which were cultured on primary culture plates as stated in the manufacturer's instructions. With the use of specific primers used in prior researches, PCR analysis was done to detect virulence genes (iucB, iutA, iroN, kfu, allS, fimH, ybtS, mrkD, and entB) (Table 1), and the results were authenticated by sequencing. A total volume of 25 μl reaction solution was used for PCR which contained 12.5 μl of 2× Master Mix (SinaClon, Iran, CAT. No., PR901638), 1 μl of 10 pmol of each primer, 2 μl (20 ng) of DNA template, and 8.5 μl of sterile distilled water. The amplification reactions were done by thermal cycler (Eppendorf, MasterCycler Gradient, Germany). In the first phase, denaturation at 94°C for 5 min was done. Then, 36 cycles of denaturation at 94°C for 45 s were performed, and in the next stage, annealing at 50-60°C for 45 s was done. Then, extension at 72°C for 45 s and a final extension at 72°C for 5 min were carried out [13 ].
The electrophorese of PCR products was done by 1–1.5% agarose gel; then, they were visualized by the use of DNA SafeStain (SinaClon, Tehran, Iran), and in the next stage, they were photographed under UV light. Positive control isolates were generously provided.

The specific primers for hippuricase (hipO), glycine (gly), CIDIF, and phospholipase C (plc) genes were used for the identification of C. jejuni, C. coli, C. difficile, and C. perfringens, respectively. The polymerase chain reaction (PCR) was optimized using the four-pair primer sets listed in Table 1. The PCR amplification was settled in 25 μl reaction volumes on a thermal cycler (Eppendorf Mastercycler Gradient, Eppendorf, Hamburg, Germany). The reaction mixture contained 10 μl master mix (Amplicon, Denmark), 0.5 μM of each primer, 1 μl DNA template, and 10 μl distilled water. The optimal PCR cycles and thermal conditions are mentioned in Table 1. PCR products were screened on a 1–1.5% agarose gel, visualized by DNA-safe stain (SinaClon Co., Iran), and photographed under UV light.

The presence of biofilm-formation genes, including smf-1, rmlA, spgM, and rpfF, along with the antibiotic-resistance genes, including L1, L2, sul1, sul2, sul3, dfrA13, and Smqnr, was assessed by PCR using the primers and conditions indicated in Table 1. Amplifications were carried out in a thermocycler (Eppendorf, Master Cycler Gradient, Germany). PCR products were analyzed by electrophoresis on 1.5% agarose gel in TBE buffer, stained with DNA-safe stain (SinaClon-Iran), and then visualization under the UV transilluminator.

All the PCR samples were tested for the presence of 16S ribosomal subunit and vapA genes to confirm the presence of R. equi DNA. Nucleotide sequences of primers include 16S forward, TCG TCC GTG AAA ACT TGG G; 16S reverse, CGA CCA CAA GGG GGC CGT; VP forward, GGT TCT CGT AAC GCT ACA ATC and VP reverse, GGT TCG TCT TTC TGA AGG TT (Sellon et al., 2001 (link)). Each PCR mixture contained 5 μl of DNA, 5 pmol of each primer and 2.5 U of PCR Master Mix 2x (Cat No. MM2011, Sinacolon), making up a total volume of 25 μl. The first series of PCR reactions involved DNA denaturation at 94°C for 2 min followed by 35 amplification cycles, each comprising of denaturation at 94°C for 35 s, primer annealing at 64°C for 30 s and extension at 72°C for 1 min. Each test run included positive (R. equi strain microbial collection of Mr. Sharghi Bacterial Laboratory) and negative controls. Finally, 1.5% agarose gel was stained with DNA Safe Stain (cat No. EP5082, Sinaclon) and electrophoresed with 85 volts for 90 min in 1X Tris Borate EDTA (TBE) buffer to read the PCR results.

Genomic DNA was extracted from fecal samples using the DNA extraction kit (AllPrep DNA minikit (Qiagen, Inc.) according to the manufacturer’s guidelines, and each DNA sample was eluted in 200 ml buffer preserved at − 80 °C until further use. Polymerase chain reaction (PCR) was conducted to detect T. gondii using specific primer pairs including F: 5′-GTAGCGTGCTTGTTGGCGAC-3′ and R: 5′-ACAAGACATAGAGTGCCCC-3′.
PCR was conducted at a final volume of 25 μl including 0.5 μl of 10 mM of each deoxynucleoside triphosphate (dNTPs), 3 μl of 10× PCR buffer without MgCl2, 2.5 mmol/l MgCl2, 1 unit of Taq polymerase (Cinnagene, Iran), 0.5 μM of each primer (10 mM), 3 μl of template DNA, and 7.5 μL of sterile distilled water. Amplification reactions were performed under the following condition: one cycle at 95 °C for 4 min, followed by 36 cycles at 94 °C for 45 s, annealing at 56 °C for 45 s, and preservation at 72 °C for 1 min with the final extension step at 72 °C for 10 min following the last cycle. PCR products were screened on a 1–1.5% agarose gel, visualized by DNA safe stain (SinaClon Co., Iran), and photographed under UV light. Moreover, PCR-amplified products were confirmed by sequencing analysis (Macrogen Korea), and the obtained sequence results were examined by using the NCBI BLAST program (Primer blast).

Total genomic DNA extraction was performed with the Cetyltrimethylammonium Bromide (CTAB) procedure as described by Nahaei et al. [16 (link)]. For final confirmation of methicillin-resistant S. epidermidis (MRSE) and methicillin-resistant S. haemolyticus (MRSH), the PCR method targeted the mecA gene was used. PCR conditions, the volume of materials, and primer sequence were set based on a previously published study by de Allori et al. [22 (link)]. Moreover, PCR analysis for icaA and icaD genes was performed using specific primers. The volume of materials, PCR conditions, and primer sequences have been described previously by Arciola et al. [23 (link)]. The PCR products were electrophoresed on 1.5% agarose gels in TBE buffer (89 mM Tris base, 89 mM boronic acid, 2 mM Na2, EDTA, pH 8.25). The gel was stained using the DNA-safe stain (SinaClon Co., Iran) and was observed under ultraviolet light. A 100 bp DNA ladder was used as a molecular size indicator.