Microbiological analysis
PD
To obtain samples of subgingival microbiota from the periodontal pocket, sterilized #15 paper points were inserted in the gingival sulcus of two maxillary teeth (#16 and #21) and two mandibular teeth (#36 and #41) of subjects at a pocket depth (PD) of less than 4 mm for 10 s and then placed in a sterilized 1.5 ml tube. They were frozen at − 20 °C until just before analysis. DNA was extracted from the collected #15 paper points using the AccuPrep Universal RNA Extraction Kit (Bioneer, Daejeon, Korea). The extraction was performed according to the manufacturer’s instructions. OligoMix (YD Global Life Science Co., Ltd., Seongnam, Korea) and three oligonucleotides (forward primer, reverse primer, and probe) (Table Primers and probes used in the real-time PCR assays5′-GAGGAATACCGGTGGCGAAG-3’5′-GGCACCGAGATTTGACTCCC-3’5′-FAM-GGTACGAAAGCGTGGGGAGCA-BHQ1-3’5′-GCGCAAGTAACGAAAGCAAAA-3’5′-GATTTTGCGCCACACTCGTT-3’5′-FAM-TGCTGCACCTAAAACAGACGACACA-BHQ1-3’5′-TGCTTGCCGGTGACTTAGGA-3’5′-AAACCGGGCTCAACAACCAT-3’5′-Texas Red-TTGAGGAGCCGGTGACTTTGG-BHQ2-3’5′-ACACGGTGTATCGTGACGGC-3’5′-GCCGGCTGCGTACTTAACCT-3’5′-HEX-CGACCTACCGCGATGCAGGA-BHQ1-3’5′-AGAAAGGCTTTGGGCGACAG-3’5′-GCTGGAGCCGTAGCTTCCAT-3’5′-Cy5-CGGGTCCTCACCCGCTCTTC-BHQ2-3’5′-GGCTGTCGTCAGCTCGTGTC-3’5′-CTCATCGCAGGCAGTATCGC-3’5′-FAM-AACGAGCGCAACCCCTTTCG-BHQ1-3’5′-CACACGCTGGCGAAACCTAC-3’5′-CACGTGGCGTTGCTTCTTTC-3’5′-HEX-CCGAAGATGCGCCGTTGAAC-BHQ1-3’5′-AGCAAGCTGTAGGCGAGGCT-3’5′-GCTGAACACTTTCGCGTGCT-3’5′-Texas Red-GCTCGTATTGCAGCCCGCAA-BHQ2-3’5′-GCCAACTGCTGCTGGAAGTG-3’5′-GCCGCTGATTTCGGAGAGTT-3’5′-HEX- ACAGCCATCGGCACAGGCAT-BHQ1–3’5′-AAATTTAAGCGGCGACGAGG-3’5′-TCCTTGCTCACGCTTACGGA-3’5′-HEX-GGCTTTGACGCGGGCGTAGT-BHQ1-3’