Faecal sample collection, DNA isolation and gut microbe quantification
LYSIS
Participants were provided with a stool sample collection kit at the baseline and the end of the study. Each kit contained a container, an anaerobic bag with anaerobic sachet plus a freezer pack. Samples were delivered to the HNRU and stored at −80 °C. Bacterial DNA was extracted using an Isolate Faecal DNA kit (Bioline, NSW, Australia) at the completion of the study. Briefly, 250 μl of each homogenised faecal sample was mixed with 750 μl of lysis buffer in a tube containing bashing beads and processed with a beater at a maximum speed for 10 min to disrupt the cells following the manufacturer's instructions. The faecal DNA was eluted from the column and stored at −80 °C for later analysis. The concentration and purity of extracted DNA were measured using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, USA). The quantitative real-time polymerase chain reaction (qRT-PCR), using SYBR™ Green Master Mix, was performed on a LightCycler® 480 Real-Time PCR instrument (Roche Applied Science). The specific primers for bacteria are presented in Primers used for quantitative real-time polymerase chain reaction (qRT-PCR)