Conventional qualitative PCR was performed to evaluate the isolates of the fecal samples retrieved in the positive fraction of the magnetic separation. To detect pks+ bacterial DNA, primers amplifying a region of the clbB gene were used following the previously published protocols and primers (forward primer 5′-GATTTGGATACTGGCGATAACCG-3′ and reverse primer 5′-CCATTTCCCGTTTGAGCACAC-3′) (2 (link), 47 (link)). About 20 mL of reaction mixture for conventional qualitative PCR consisted of 1 µL of DNA (DNA concentrations are summarized in Table S5 ), each 0.6 µL of 10 µmol/L forward and reverse primer solutions, deoxynucleotide mixture, the DNA polymerase Master Mix RED (Ampliqon), and H2O. The PCR conditions were 10 min at 95°C, 30 cycles of 45 s at 95°C, 45 s at 54°C, and 1 min at 72°C, and a final step of 7 min at 72°C. E. coli Nissle 1917 was selected as the positive control for pks+ detection and E. coli LMG2092 as the negative control.
Dna polymerase master mix red
Manufactured by Ampliqon
Sourced in Denmark
DNA Polymerase Master Mix RED is a ready-to-use solution containing all the necessary components for performing PCR amplification, including DNA polymerase, nucleotides, and buffer. It is designed to provide consistent and reliable results in PCR experiments.
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Lab products found in correlation
4 protocols using dna polymerase master mix red
1
Detection of pks+ Bacterial DNA
2
Rapid ESBL Gene Detection by Boiling PCR
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The boiling method was used to prepare template DNA for PCR as described by Qi et al. 16 . The oligonucleotide primers used to detect bla CTX-M group 1-4 , bla TEM , bla SHV , bla OXA , bla PER , bla KPC , and bla NDM are listed in Table 1 [17] [18] [19] [20] . PCR amplification was performed in a total volume of 50µL containing 1µL of each primer (10pM), 25µL of DNA Polymerase Master Mix RED (Ampliqon, Co., Denmark), 2µL of DNA, and 21µL of DNase and RNase free water in a FlexCycler PCR Thermal Cycler (Analytik Jena, Germany) under the following cycling conditions: an initial denaturation step at 95°C for 5min followed by 30 cycles of denaturation at 95 °C for 1min, annealing at 55-61°C for 1min (Table 1), extension at 72°C for 1min, and a final extension step at 72°C for 5min. The PCR products were separated by electrophoresis in a 1.5% agarose gel in 0.5 Tris, EDTA, Boric acid (TBE) buffer. For each ESBL group, amplicons from randomized selected strains were sequenced (Macrogen, South Korea). The obtained nucleotide sequences were compared with those in the GenBank database for homologous nucleotide sequences by BLAST (www.ncbi.nih.gov/BLAST program).
3
Detection of Carbapenemase-Encoding Genes
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DNA templates were extracted by a genomic DNA extraction kit (Bioneer, Daejeon, Korea) according to the manufacturer’s instructions. The primers used in this study have been listed in Table 1 . The following carbapenemase-encoding genes were detected: class A β-lactamase gene: blaKPC, class B MBL genes: blaIMP, blaVIM, blaSPM, blaGIM, blaSIM, blaGES, and blaNDM, class D oxacillinases genes: blaOXA-23-like, blaOXA-24like, blaOXA-51-like, and blaOXA-58-like, and IS elements such as ISAba1, ISAba2, ISAba3, ISAba4, IS18, ISAba1–blaOXA-51-like, and ISAba1–blaOXA-23-like. PCR amplification was performed in a total volume of 25 µl containing 0.5 µl of each primer (10 pM), 12.5 µl of DNA polymerase master mix RED (Ampliqon A/S, Odense, Denmark), 1 µl of DNA, and 10.5 µl of water (DNase and RNase free water). The PCR cycle consisted of denaturation at 94°C for 5 min followed by 35 cycles at 94°C for 30 s, annealing at 51 – 59°C for 40 s, and extension at 72°C for 40 s.
4
Molecular Detection of Carbapenemase Genes
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The boiling method was applied to extract the template DNA to detect carbapenemase genes. The primers were used to detect blaKPC, blaOXA-48-like, blaIMP, blaVIM, and blaNDM as described by Doyle et al.5 (link)
The amplification reaction was carried out in a final volume of 25 μL containing 0.5 μL of each primer (10 pM), 12.5 μL of DNA Polymerase Master Mix RED (Ampliqon, Co., Denmark), 1 μL of DNA, and 10.5 μL of DNase and RNase-free water in a FlexCycler PCR. The Thermal Cycler (Analytik Jena, Germany) was performed through the following cycling conditions: an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of DNA denaturation at 95 °C for 45 s, annealing at 45 s, and extension at 72 °C for 1 min, and a final extension at 72 °C for 8 min.
The PCR products were analyzed with gel electrophoresis on a 1.5% agarose gel in 0.5 Tris, EDTA, and Boric acid (TBE) buffer, followed by staining with KBC stain (CinnaGen, Iran) and visualized using a UV transilluminator. Positive controls for these genes were obtained from Pasteur Institute of Iran, Tehran.
The amplification reaction was carried out in a final volume of 25 μL containing 0.5 μL of each primer (10 pM), 12.5 μL of DNA Polymerase Master Mix RED (Ampliqon, Co., Denmark), 1 μL of DNA, and 10.5 μL of DNase and RNase-free water in a FlexCycler PCR. The Thermal Cycler (Analytik Jena, Germany) was performed through the following cycling conditions: an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of DNA denaturation at 95 °C for 45 s, annealing at 45 s, and extension at 72 °C for 1 min, and a final extension at 72 °C for 8 min.
The PCR products were analyzed with gel electrophoresis on a 1.5% agarose gel in 0.5 Tris, EDTA, and Boric acid (TBE) buffer, followed by staining with KBC stain (CinnaGen, Iran) and visualized using a UV transilluminator. Positive controls for these genes were obtained from Pasteur Institute of Iran, Tehran.
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