The BOXA1R primer (5′-CTACGGCAAGGCGACGCTGACG-3′) (47 (link)) was used for BOX-PCR targeting the BOX-repetitive element (51 , 82 ). The PCR amplification of DNA was performed in 50 μL containing 100 μM of the BOXA1 primer, 24 μL Dream Taq Green 2× Master Mix (Thermo Fisher), 24 μL nuclease-free water, and the DNA extract. PCR amplifications were performed in a thermal cycler (ICYCLER Thermal Cycler, Bio-Rad Laboratories, Hercules, CA, USA) with an initial denaturation (95°C, 2 min) followed by 35 cycles of denaturation (92°C, 30 s), annealing (50°C, 1 min), and extension (65°C, 8 min) with a final extension (65°C, 8 min). Each PCR product was analyzed by electrophoresis on 1.5% agarose gels. DNA extracted from Rhizobium leguminosarum strain RhPs 10 from the microbial strain collection of the Institute for Soil Sciences and Agricultural Chemistry (30 ) was used as a positive control.
Dreamtaq green 2 master mix
Manufactured by Thermo Fisher Scientific
DreamTaq Green 2× Master Mix is a ready-to-use solution for PCR amplification. It contains DreamTaq DNA Polymerase, dNTPs, and an optimized buffer system, along with a tracking dye and a density reagent for direct gel loading.
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4 protocols using dreamtaq green 2 master mix
1
BOX-PCR Amplification of Rhizobium leguminosarum
2
mcr-1 gene detection in E. coli
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Direct colony PCR was carried out on 100 randomly selected E. coli strains each from the 2019 to 2020 collections, respectively. This became necessary as the mcr‐1 gene has often been shown to exist unexpressed (Fernandes et al., 2016 (link); Hassan et al., 2021 (link); Lentz et al., 2016 ; Terveer et al., 2017 (link)). For each year of collection, 50 strains were selected per source, that is chicken carcasses and faeces. The online research randomizer (version 4.0) computer software was utilized for the selection as all strains were serially numbered. The programme generated random numbers identifying strains to be included. Colonies served as DNA template in the reaction mixtures, with 12.5 μL of DreamTaq Green 2× Master Mix (Thermo Fisher Scientific), 6.5 μL nuclease‐free water and 0.5 μL of each primer solution (F: 5′CGGTCAGTCCGTTTGTTC3′ and R: 5′CTTGGTCGGTCTGTAGGG3′) specific for the mcr‐1 gene. The thermo‐cycling condition was maintained at 94°C 15 min + 25 × (94°C 30 s + 58°C 90 s + 72°C 60 s) + 72°C 10 min, using a MiniAmp Plus Thermal Cycler (Thermo Fisher Scientific). Resulting amplicons were subjected to agarose gel electrophoresis using 1.5% gel in 1× TBE with ethidium bromide. The gels ran for 90 min at 90 V before visualizing the bands (Cavaco et al., 2016 ). The PCR product expected for the mcr‐1 gene was expected to have 309 base pairs.
3
Extracting and Amplifying MDV DNA
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DNA was extracted from infected FFE cells scraped from FFs or CEC cultures using the QIAamp DNA Mini Kit from Qiagen, LLC (Germantown, MD, USA) according to the manufacturer’s instructions. PCR was performed using DreamTaq Green 2× Master Mix (Thermo Fisher Scientific) and previously described primers to MDV gC and UL13 [23 (link),24 (link)]. All PCR reactions were electrophoresed through 0.8% agarose gels and recorded using an Alpha Imager HP (Protein Simple) (Wallingford, CT, USA).
4
Fungal ITS and EF1α Amplification
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Internal transcribed spacer (ITS) regions surrounding the 5.8S rRNA gene were amplified with primers ITS1 and ITS4 (White et al. 1990 ). For the PCR reaction, DreamTaq Green 2× Master Mix (Thermo Scientific) was used with 10 µM of each primer and 50 to 100 ng of template DNA. The 50 µl reaction volumes were completed with PCRgrade water. The following program was used during the PCR procedure (PTC-200, MJ Research): initial denaturation at 95°C for 3 min, 35 amplification cycles (95°C for 30 s, 52°C for 30 s and 72°C for 45 s) and final elongation at 72°C for 10 min. An additional PCR-reaction was conducted using primer pair EF1αF and EF1αR to amplify the translation elongation factor 1 alpha (EF1α) gene, according to Geiser et al. (2004) (link). Reaction conditions were as described above, except for an an nealing temperature of 53°C.
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