The presence of classical enterotoxin genes, sea, seb, sec, sed, and see (Table 1 ; Barati et al., 2006 ) among the Staphylococcus isolates obtained, was investigated employing a multiplex PCR assay as previously described by Omoe et al. (2005 (link)). The reaction mixture (50 μl) containing 0.1 µM of each primer, 50–100 ng genomic DNA, and 25 μl of Taq DNA polymerase 2.0× Master Mix RED was used (1.5 mM MgCl2; Ampliqon). Staph. aureus reference strains, Staph. aureus DSM 19,040 (SEC, SEE) and Staph. aureus DSM 19,041 (SEA, SEB, SED), were used as enterotoxin producers (Rahmdel et al., 2018 (link)). The products of PCR were detected by electrophoresis in a 1.4% agarose gel in TAE (1×).
Taq dna polymerase 2.0 master mix red
Manufactured by Ampliqon
Sourced in Denmark
Taq DNA polymerase 2.0× Master Mix RED is a pre-mixed solution containing Taq DNA polymerase, PCR buffer, MgCl2, and dNTPs. It is designed for routine PCR applications.
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Lab products found in correlation
3 protocols using taq dna polymerase 2.0 master mix red
1
Multiplex PCR for Staphylococcus Enterotoxins
2
Genetic Profiling of S. epidermidis Pathogenicity
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S. epidermidis TYH1 WT and ΔhdcA mutant strains were examined for the presence of genes coding pathogenicity factors, including classical enterotoxin genes sea, seb, sec, sed, see, enterotoxin-like toxin Q gene (selq), and TSST-1 gene (tst1) (12 (link)), coagulase (13 (link)), and nuclease (14 (link)). Table 1 shows the PCR primer pairs. PCR amplifications were performed as described by Rahmdel et al. (15 (link)). For selq, tst1, coa and nuc detection, the uniplex PCR assay using each primer pair was applied. The reaction mixture (25 μL) contained 12.5 μL of Taq DNA Polymerase 2.0× Master Mix RED (1.5mM MgCl2; Ampliqon, Copenhagen, Denmark), 0.4 μM of each primer, and 50–100 ng DNA template. Superantigenic toxin genes, (SEs), were co-amplified by multiplex PCR according to Omoe et al. (16 (link)). The positive control strains used in this study were S. aureus DSM 19040 (sec, see), and S. aureus DSM 19041 (sea, seb, and sed).
3
Genomic DNA Extraction and Amplification
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Genomic DNA was extracted from clinical samples using QIAamp® DNA Mini and Blood Mini kits (Qiagen, Düsseldorf, Germany). DNA amplification was performed in PCR reaction mixture consisting of genomic DNA (5 μl), Taq DNA polymerase 2.0 Master Mix Red (MgCl2, 1.5 mM; 25 μl; Ampliqon, Glostrup, Denmark), the first set of primers (10 μM, 0.25 μl each; Table I ) and water (19.5 μl). PCR was carried out for 35 cycles under the following conditions: Initial denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min, followed by a final extension step at 72°C for 10 min.
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