DNA extraction was performed using the EZ1®DNA tissue kit (Qiagen, Hiden, Germany) on BIOROBOT EZ1 (Qiagen, Hiden, Germany), according to the manufacturer’s instructions. Initially, we mixed in tubes about 200 mg of stool with 360 µL of G2 lysis buffer (Qiagen, Hiden, Germany). This was mechanically lysed with tungsten beads (Qiagen, Hiden, Germany) using FastPrep-24TM 5G Grinder for 40 s. After 10 min of incubation at 100 °C to allow for complete lysis, tubes were centrifuged at 10,000× g for 1 min. Subsequently, 200 μL of supernatant was enzymatically digested using 20 μL of proteinase K (20 mg/mL, Qiagen) and incubated overnight at 56 °C. DNA was extracted from 200 µL of sample, eluted in 200 µL volume, then aliquoted in individual tubes of: pure extracted DNA, diluted to 1:10 and finally DNA diluted to 1:100.
To control the extraction quality and the absence of PCR inhibitors, universal eubacterial qPCR targeting the 16S rRNA bacterial genes, named “qPCR all bacteria” [70 (link)], was performed on pure DNA, dilutions to1:10 and to 1:100. By comparison of Ct values obtained for pure and diluted DNAs, the dilution to1:10 were chosen for parasite screening. DNA tubes were stored at −20 °C until use.
To control the extraction quality and the absence of PCR inhibitors, universal eubacterial qPCR targeting the 16S rRNA bacterial genes, named “qPCR all bacteria” [70 (link)], was performed on pure DNA, dilutions to1:10 and to 1:100. By comparison of Ct values obtained for pure and diluted DNAs, the dilution to1:10 were chosen for parasite screening. DNA tubes were stored at −20 °C until use.