Bacterial Strains and Growth Conditions
In this study, L. monocytogenes ATCC 19115 and 47 clinical L. monocytogenes strains from the Microbial bank of Department of Microbiology affiliated to Iran University of Medical Sciences, Tehran, Iran, were used (Table 1) . Isolates were cultured on Brain Heart Infusion (BHI) agar (Merck, Darmstadt, Germany) at 37°C for 48 h. DNA of the samples was extracted with a DNA Extraction kit (Roche, Germany) according to the manufacturer’s protocol. DNA purity and concentration were assessed using a NanoDrop Spectrophotometer (Thermo Scientific, USA).
In this study, we used 5 VNTR loci for MLVA-HRMA based on the previously designed primers . The PCR amplification and HRMA were performed using a Rotor-Gene thermal cycler (Corbett Life Sciences, Sydney, Australia). The PCR reaction solution was performed with a total of 20 µL, containing 1 µL of each forward and reverse primers, 1 μL of template DNA (0.5 μg), 4 μl of 5× Hot Firepol Eva Green HRM Mix (Solis BioDyne), and 13 μL of sterile distilled water.
The thermal program was performed by an initial denaturation at 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s, 54 °C for 22 s, and 72 °C for 40 s for VNTR locus 1 (Lm_10), 95 °C for 30 s, 52 °C for 22 s, and 72 °C for 40 s for VNTR locus 2 (Lm_11), 95 °C for 30 s, 55.5 °C for 30 s, and 72 °C for 30 s for VNTR locus 3 (Lm_23), 95 °C for 30 s, 54 °C for 30 s, 72 °C for 45 s for VNTR locus 4 (Lm_32) and 95 °C for 30 s, 57 °C for 22 s, and 72 °C for 40 s for VNTR locus 5 (LM_TR6).
After PCR amplification, the HRM step was performed. For HRM step the fluorescence was measured by increasing the temperature from 75° to 95°C with a rate of 0.1 °C/s. In case of nucleotide changes in different strains, various curves are produced.
The melting curve and different plots of strain 6 were used as the baseline control. Each strain with equal waveform was grouped. Then, two strains were randomly selected from each group and the PCR reaction was performed for the studied genes using a DNA thermal cycler (PeqLab, Germany) with the following proﬁle: initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 22 s, and extension at 72°C for 40 s, with a final extension step at 72°C for 5 min. The PCR product was sent for sequencing to Takapozist Company, Iran (on behalf of Bioneer Company, Korea). The allele number was determined and imputed for each locus into BioNumerics (Applied Maths, Sint-Martens-Latem, Belgium) to draw the UPGMA dendrogram and the minimum spanning tree for all the studied L. monocytogenes.