DNA Extraction for 16S Amplicon Analysis

All the procedures mentioned here were performed under sterile conditions. Before extracting the DNA, intestine samples were transferred to a sterile Petri dish and placed on a cool-pack on dry ice. The intestine was opened and transferred to a 5ml tube containing 1.4mm Zirconium oxide beads (Cayman Chemical, Ann Arbor, Michigan, United States) and 2ml of InhibitEX buffer (Qiagen). Thereafter, DNA was extracted immediately using QIAamp DNA stool Mini Kit (Qiagen) according to the manufacturer’s protocol. The final elution volume was 75μl (ATE buffer). The same extraction method was employed for the mouth samples. The quality and quantity of the extracted DNA were checked with NanoDrop spectrophotometer ND-8000 (ThermoFisher Scientific).