Qiaamp dna stool mini

DNA was extracted using the QIAamp DNA Stool Mini (QIAGEN, Hilden, Germany) following the manufacturer’s recommendations. The 16S rRNA V3/V4 region was amplified using primers 341F (5′-CCTACGGGRSGCAGCAG-3’) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with Illumina adapters for sequencing. Amplification was performed in 35 cycles at an annealing temperature of 50°C, and analysis was conducted in triplicate. Sequencing was performed on an Illumina MiSeq platform using the V2-500 kit with a 500 bp paired-end run.

DNA was extracted from stool samples using the QIAamp DNA Stool Mini kit protocol (Qiagen, Germany). During DNA extraction, four fecal samples from GDM patients and four fecal samples from healthy subjects were excluded, which changed the sample size from 27 to 23 in the GDM group and from 30 to 26 in the healthy group.

Stool samples were collected from family dogs during the scheduled appointments immediately after spontaneous defecation using a faecal swab (Fecal Swab™, Copan Diagnostics Inc., USA) inserted in the stool, being careful not to touch the soil, grass or surrounding items. Fresh samples were refrigerated at 4 °C and shipped to Laboratory of Microbial Ecology and Genomics at the Istituto Zooprofilattico Sperimentale delle Venezie (Legnaro, Padua) within 24 h, where they were processed as soon as received. Two total DNA extractions were performed by column-based kit QIAamp DNA Stool Mini (Qiagen, USA) for each sample in order to guarantee better representativeness of the entire microbial community. The extracted bacterial DNA was preserved at − 80 °C.