Nzy soil gdna isolation kit

Fecal samples (1–3 g) were collected from both groups before (D0) and after 1 month on the MCT diet (D30) directly from the rectal ampoule with sterile gloves and immediately frozen at −80 °C to fix bacterial growth and preserve DNA content.
Bacterial DNA extraction from fecal samples and sequencing of bacterial 16S rRNA gene procedures were the same as we previously published [28 (link)]. Bacterial DNA was extracted from fecal samples using the NZY Soil gDNA Isolation kit (NZYTech, Lisboa, Portugal). The V4 region of the 16S rRNA gene was amplified using specific primers (515F-806R) [29 (link)] with a barcode. All PCR reactions were carried out with Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA). Sequencing libraries were generated using NEBNext Ultra DNA Library Pre^® Kit for Illumina^® (New England Biolabs, Ipswich, MA, USA). The library was then sequenced on an Illumina MiSeq platform and 250 bp paired-end reads were generated.

Approximately 250–300 mg from each of the 192 stool samples was used for DNA extraction using the NZY Soil gDNA Isolation Kit (NZYTECH, Lisbon, Portugal) following the manufacturers’ instructions. Eluted and purified DNA (50 µl) was aliquoted in two vials of the same volume and stored at − 20 ºC until use. Once DNA samples were used for molecular field analysis by LAMP at Nossa Senhora da Paz Hospital, they were stored again at − 20 ºC until they were later shipped to Centre for Research in Tropical Diseases of the University of Salamanca (CIETUS, Salamanca, Spain) for molecular reanalysis by LAMP and qPCR. DNA was extracted and stored as stool samples were collected, so that the first DNA samples obtained were stored for longer (approximately 3 months) than those obtained at the end of the study (approximately 3 weeks) before being shipped to our laboratory in Spain. The DNA samples were kept frozen whenever possible because of possible power outages at the Nossa Senhora da Paz Hospital. Besides, it was not possible to maintain the cold chain during the entire transport of the samples to Spain.

Approximately 250–300 mg from each of the 192 stool samples was used for DNA extraction using the NZY Soil gDNA Isolation Kit (NZYTECH, Lisbon, Portugal) following the manufacturers’ instructions. Eluted and purified DNA (50 µl) was aliquoted in two vials of the same volume and stored at − 20 ºC until use. Once DNA samples were used for molecular field analysis by LAMP at Nossa Senhora da Paz Hospital, they were stored again at − 20 ºC until they were later shipped to Centre for Research in Tropical Diseases of the University of Salamanca (CIETUS, Salamanca, Spain) for molecular reanalysis by LAMP and qPCR. DNA was extracted and stored as stool samples were collected, so that the first DNA samples obtained were stored for longer (approximately 3 months) than those obtained at the end of the study (approximately 3 weeks) before being shipped to our laboratory in Spain. The DNA samples were kept frozen whenever possible because of possible power outages at the Nossa Senhora da Paz Hospital. Besides, it was not possible to maintain the cold chain during the entire transport of the samples to Spain.