Lysis solution f

The contents of the small intestine were combined with Lysis Solution F (Nippon Gene, Toyama, Japan), crushed using a Shake Master Neo (BMS, Waseda, Japan), and incubated at 65 °C for 10 min. DNA was extracted from the solution using a Lab-Aid824s DNA Extraction kit (ZEESAN Biotech, Xiamen, China). Libraries were generated using a 2-step tailed PCR method, and the amplicons were sequenced on a MiSeq (Illumina, San Diego, CA, USA). The 16S rRNA reads were processed with QIIME2 (version: 2023.2). After trimming low-quality and chimeric reads, the feature-classifier plugin was utilized to compare the obtained representative sequences with the EzBioCloud 16S database for phylogenetic inference. Alignment and phylogeny plugins were then used to construct phylogenetic trees. Alpha and beta diversity analyses were conducted using the diversity plugin of QIIME2.

The collected dentin shavings were freeze-dried (VD-250R Freeze Dryer, TAITEC, Saitama, Japan) and pulverized at 1500 rpm for 2 min (Shake Master Neo, Bio Medical Science, Tokyo, Japan). The powdered root caries samples were added to a lysis solution (Lysis Solution F, Nippon Gene, Tokyo, Japan) and left to stand for 10 min at 65 °C. The samples were centrifuged at 12,000× g for 1 min, and genomic DNA was extracted using an MPure Bacterial DNA Extraction Kit (MP Biomedical, Santa Ana, CA, USA).

Faecal samples were collected using a guanidine thiocyanate solution (Faeces Collection kit; Techno Suruga Lab, Shizuoka, Japan). After vigorous mixing, the samples were stored at 4 °C for a maximum of 7 days until DNA extraction. After homogenisation with lysis solution F (Nippon Gene, Tokyo, Japan), the genomic DNA was heated at 65 °C for 10 min and purified from the supernatants using the MPure Bacterial DNA Extraction Kit (MP Biomedicals, Solon, OH, USA) with MPure-12 system (MP Biomedicals). The purified DNA was quantified using Synergy LX (BioTek Instruments, Winooski, VT, USA) and QuantiFluor system (Promega, Madison, WI, USA).