Five healthy volunteers participated the study, two males and three females respectively. The five volunteers did not treat with antibiotics in the past three months. The volunteers were required to not eat food or drink water at least one hour before sampling. Totally, five saliva samples and five oral swab samples were collected. These samples were named as Sa1, Sa2, Sa3, Sa4, Sa5, Os1, Os2, Os3, Os4, Os5, respectively. The genomic DNA was extracted using QIAmp® DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Extracted DNA was quantified by Nanodrop™ 2000 spectrophotometer (Thermo Scientific,Wilmington, NC, USA). Forward primer and reverse primer were 518 F CCAGCAGCCGCGGTAAT and 806R GGACTACCAGGGTATCTAATCCTGTT respectively, targeting at 16S rRNA V4 region of bacteria [
Oral microbiota: microbial biomarkers of metabolic syndrome independent of host genetic factors.
]. The reaction mixture with a final volume of 10 μl contained: 0.2 μM forward primer and reverse primer, 10 ng genomic DNA, 5 μl 2×Light Cycler® 480 High Resolution Melting Master Mix (Roche, Mannheim, Germany), 1.2 μl 25 Mm MgCl2
and DNase/RNase free water. The touch down PCR and High-resolution melting analysis was performed on the Light Cycler 480(Roche, Mannheim, Germany). The condition of touch down PCR was 95℃ for 10 min, 55 cycles of 94℃ for 10 s, annealing at 65℃ for 10 s with a 0.5℃ decrease in temperature after each cycle down to 53℃, and extension at 72℃ for 10 s. HRM analysis was performed at the end of PCR protocol under the following condition: 95℃ for 1 min, 40℃ for 1 min, temperature continuously increased from 75℃ to 95℃ at a 0.2℃/s to acquire sample fluorescence, 40℃ for 10 s to cool. The result of the HRM was analyzed according to the Light Cycler 480 software.