Rectal swabs were thawed, and DNA was extracted using a QIAmp DNA Stool kit (Qiagen, Hilden, Germany) according to the manufacturer instructions with a minor modification. The rectal swabs were dissolved in 1 ml Buffer ASL and shaken at 1,000 rpm (Mixing Block MB-102, CaRlibiotech S.r.l. Rome, Italy) continuously until the stool samples were homogenized. DNA quality and quantity were assessed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and the isolated DNA was stored at −20°C until use.
Bacterial DNA was amplified by targeting the V3–V4 hypervariable regions of the 16S rRNA gene (23 (link)). PCR amplification of each sample was performed in a 25-μl volume. A total of 12.5 μl of KAPA HIFI Master Mix 2× (Kapa Biosystems, Inc., MA, USA) were used. Then, 0.2 μl of each primer (100 μM) was added to 2 μl of genomic DNA (5 ng/μl). Blank controls (no DNA template) were also included. Amplification and library quantification were carried out as described previously (24 (link)).
Bacterial DNA was amplified by targeting the V3–V4 hypervariable regions of the 16S rRNA gene (23 (link)). PCR amplification of each sample was performed in a 25-μl volume. A total of 12.5 μl of KAPA HIFI Master Mix 2× (Kapa Biosystems, Inc., MA, USA) were used. Then, 0.2 μl of each primer (100 μM) was added to 2 μl of genomic DNA (5 ng/μl). Blank controls (no DNA template) were also included. Amplification and library quantification were carried out as described previously (24 (link)).
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