Opticon monitor analysis software version 3

All data generated by qPCR were analyzed by the Opticon Monitor Analysis Software version 3.4 (Biorad, Milan, Italy). To quantify the effect of PMA on heat-killed cells, Δ log values were calculated (log value qPCR − log value PMA-qPCR).
The statistical analysis was conducted with the statistical package IBM SPSS Statistics 24.0 (IBM Corporation, Armork, NY, USA). Significant differences between the doses of PMA tested for cytotoxic effect and between the two exposure protocols were assessed by ANOVA, Tukey’s multiple comparison, and a t-test.

The qPCR data were analysed using Opticon Monitor Analysis Software version 3.4 (Bio-Rad, Hercules, CA, USA). The proportions were compared using Fisher’s exact test. The Mann-Whitney U test was adopted to evaluate the between-sample (tap water vs. DUWLs) differences in the viable cell counts of Legionella and in the TVC data. Correlation was evaluated using the Pearson test.

The extracted genomic DNA was analyzed for the presence of amplifiable sequences using three qPCR commercial kits for the detection of H. pylori, S. oralis, and S. mutans according to the manufacturer’s instructions (Primerdesign^TM Ltd.) (Camberley, UK). The kit for H. pylori detection contains the reagents to amplify and quantify a 100-bp fragment of the gene segment coding for the beta subunit of RNA polymerase (rpoB), the kit for S. oralis amplifies a segment of the glucosyltransferase (gtfR) gene, and the kit for S. mutans detection amplifies a segment of the glucosyltransferase-I gene (gtfb), previously identified as a highly specific marker for S. mutans.
All the three kits provide amplification solutions containing Taq DNA polymerase, an internal control, primers, fluorescent Taqman probes, a negative control, and a positive control, the latter of which are used as standards for quantification. Under optimal PCR conditions, Primer Design detection kits can detect less than 100 copies of target template. The internal control allows the detection of any possible factors that might inhibit the amplification reaction and is amplified in the same reaction mixture; however, different primers and a probe labeled with a different fluorophore were used.
The qPCR data were analyzed using Opticon Monitor Analysis Software version 3.4 (Bio-Rad, Hercules, CA, USA).