Metapolyzyme

Details of study subject recruitment, inclusion and exclusion criteria, sampling and DNA extraction are described in a previous study [9 (link)]. In brief, whole-mouth, supragingival plaque samples were collected from 30 children, 6–10 years old, with no caries, early caries or advanced caries (n = 10, each group) recruited at the Pediatric Dentistry Clinic at the Temple University Kornberg School of Dentistry. Caries status was determined using the International Caries Classification and Management System (ICCMS) [10 (link)]. The clinical characteristics are summarized in Supplementary Table 1. For DNA extraction, the samples were first digested with Metapolyzyme (Sigma, USA) – a mixture of six enzymes, two of which (lyticase and chitinase) target the fungal cell wall. DNA was then purified using the ZymoBIOMICS Kit (Zymo Research, Germany), that involves beating with a mixture of 0.1 & 0.5 mm beads. The study was conducted in compliance with ethical standards and was approved by the Temple University’s Institutional Review Board (protocol # 24355)

750 µL of saliva from each sample was transferred to a 2-mL microcentrifuge tube and mixed with 750 µl of phosphate-buffered saline (PBS). Each tube was then centrifuged for 10 min at 15,700 ×g to pellet the cells. The supernatant was discarded and resuspended the pellet in 125 μl PBS buffer and 25 μl MetaPolyzyme (Sigma-Aldrich, Norway). After incubating the tubes at 35 °C for 4 h, oral metagenomic DNA was extracted from each saliva sample by QIAcube (Qiagen, Norway) and QIAamp® DNA Mini QIAcube Kit. A pooled metagenomic DNA sample was prepared by aliquot and mixing 10 µl of each extracted oral metagenomic DNA in a new Eppendorf tube and kept at − 20 °C.

For DNA extraction, 500 µl of each UWS sample were mixed with 500 µL PBS containing 3.75 mM dithiothreitol and then centrifuged at 13000 rpm for 10 minutes to pellet the cells. Supernatant was discarded, and the pellets were each suspended in 162 µL phosphate-buffered saline and 18 µL of Metapolyzyme (Sigma, USA) and incubated at 35°C for 4 hours for digestion. DNA was subsequently extracted using PureLink™ Genomic DNA Mini Kit (Invitrogen, USA), according to manufacturer’s instructions. DNA concentration was determined by Qubit dsDNA HS kit (Invitrogen, USA).