Molecular Identification of Thermotolerant Campylobacter

All biochemically verified isolates were subjected to PCR analysis for the detection of the 23S rRNA gene, which revealed the presence of thermotolerant Campylobacter spp., and then for the identification of the species, two differentiation genes (mapA for C. jejuni and ceuE gene for C. coli) were used. The QIAamp DNA Mini kit (Qiagen, Germany, GmbH) was used to extract DNA in accordance with the manufacturer's instructions. Briefly, 200 µl of the sample suspension was treated at 56 °C for 10 min with 10 µl of proteinase K and 200 µl of lysis solution. Then, 200 µl of 100% ethanol was added to the lysate after incubation. After that, the sample was washed and centrifuged in accordance with the manufacturer's instructions with the help of 100 µl of elution buffer, and DNA was extracted.
The oligonucleotide primers used in this study were provided by Metabion (Germany) (supplementary table 1). A 25-µl reaction containing 12.5 µl of Emerald-Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer at a 20 pmol concentration, 5.5 µl of water, and 5 µl of DNA template was used. Thermal cycler 2720 from Applied Biosystems was used to perform the reaction.
The PCR products were separated using 5 V/cm gradient electrophoresis on a 1.5% agarose gel (Applichem, Germany, GmbH) in 1 × TBE buffer at room temperature. Each gel slot had 20 µl of the product for gel analysis. The fragment sizes were calculated using the Generuler 100 bp ladder (Fermentas, Germany) and the Gelpilot 100 bp ladder (Qiagen, Gmbh, Germany). A gel documentation system (Alpha Innotech, Biometra) took pictures of the gel, and computer software was used to analyze the data.