Extraction of DNA
HEAT, LYSIS
The collected saliva sample was mixed using a vortex mixer for more than 10 s and reacted in a water bath at 50°C for 1 h. Then, 200 µL of the sample was dispensed into an Eppendorf tube (DNA LoBind Tube; Eppendorf) and allowed to react in a heat block at 75°C for 15 min. Because precipitates were identified in the sample, the sample was centrifuged at 15,000 rpm and 4°C for 10 min to separate the supernatant and precipitate. Next, 180 µL of achromopeptidase (FUJIFILM Wako Pure Chemical) was added to the precipitate and reacted at 55°C for 10 min. The supernatant and precipitates were separately treated and adsorbed onto the same column of the Virus Spin Kit (QIAGEN). After lysis with AWI Cleaning Buffer, the supernatant and precipitates were cleaned with a single column and extracted with 100 µL of UltraPureThe tongue coating sample was spiked with 1250 units of Ready‐Lyse Lysozyme Solution (Lucigen) prepared with 4 µL of TES buffer (1 mM EDTA, 10 mM Tris‐HCl [pH 7.5], and 100 mM NaCl), and incubated at 37°C overnight according to the recommendations of OMNIgene OMR‐120. The sample was then centrifuged at 15,000 rpm and 4°C for 10 min, and the supernatant and precipitates were treated in a similar manner as described above for the saliva.The concentration of DNA extracted from the saliva/tongue coating samples was measured using SimpliNano (GE Healthcare); distilled water was used as the background.