Real-time PCR
The number of oral microorganisms in saliva was measured as follows: First, participants were asked to spit approximately 1 ml of saliva into a bottle. Next, a filter paper was dipped into the bottle for 5 s to absorb the saliva. Collection was performed three times: before brushing, after brushing, and after wiping or rinsing the mouth. After saliva collection, the filter paper was placed in 500 µL PBS and allowed to stand for 30 min before DNA was collected from the samples. Real-time PCR was performed on the recovered DNA using specific primers, and the total bacterial count (Total Bacteria) was calculated from a standard curve created using synthetic DNA [