2.5. Multiplex Quantitative RT-PCR (qPCR)

The qPCR was performed with the EasyPerio molecular kit (YD Global Lifescience, Seongnam, Korea), according to the manufacturer’s instructions. The kit consisted of 8 different oligo mixes and 2 × master mixes. This was designed according to the typical multiplex qPCR method [14]. The CFX96 Touch™ RT-PCR Detection System (Bio-Rad, Hercules, CA, USA) was used for qPCR. The sequential steps in the PCR procedure were as follows: pre-denaturation for 30 s at 95 °C; 40 cycles of 5 s denaturation at 95 °C; and 30 s extension and annealing at 62 °C. Fluorescence scanning was performed after the extension and annealing step. Information on the primers and probes is displayed in Table 1. In this way, DNA of 18 species of bacteria was extracted and analyzed by RT-qPCR. The 18 species of bacteria were the following: A. actinomycetemcomitans, P. gingivalis, T. forsythia, T. denticola, Fusobacterium nucleatum, P. intermedia, Parvimonas micra, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens, Streptococcus mitis, Streptococcus mutans, Lactobacillus casei, Staphylococcus aureus, Enterococcus faecalis, Actinomyces viscosus, Prevotella nigrescens, and Streptococcus sobrinus.

Primers and probes of the 18 species of bacteria analyzed.