Dreamtaq hot start pcr master mix 2x

Total bacterial DNA from the 13 presumptive L. monocytogenes isolates was extracted using the InstaGene Matrix (BioRad Laboratories, Inc., Hercules, CA, USA) according to manufacturer’s instruction. The 16S rDNA gene was amplified by PCR and then sequenced. PCR amplifications were performed using 25 μL of DreamTaq Hot Start PCR Master Mix 2x (Thermo Scientific, Waltham, MA, USA), 0.5 μM fD1 (5′-AGAGAGTTTGATCCTGGCTCAG-3′), 0.5 μM rD2 (5′-TAAGGAGGAGGTGATCCAGCC-3′), 50–100 ng of purified DNA and 19 μL of molecular biology-grade water (Thermo Scientific). PCR mixtures were subjected to several amplification cycles, starting with an initial denaturation cycle (95 °C, 3 min), followed by 35 cycles of denaturation (95 °C, 30 s), hybridization (60 °C, 30 s), elongation (72 °C, 1 min), and ending with a final elongation cycle (72 °C, 5 min) in a thermal cycler (Eppendorf, Hamburg, Germany). The resulting amplicons were then purified using the Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel^TM, Düren, Germany) and sent to Eurofins Genomics (Ebersberg, Germany) for DNA sequencing. To determine their taxonomic identification, the nucleotide sequences were analyzed using the BLAST nucleotide server of the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/, accessed on 28 February 2024).