Quantifying Gut Microbiota Composition

Bacterial composition of total bacterial load and selected members of the GM were enumerated at the beginning t = 0 h) and after 24 h of fermentation by real-time quantitative PCR (qPCR) as previously described [21 (link)]. For gut microbiota analysis, we have selected bacteria with previously reported alterations in autism spectrum disorders [16 (link),27 (link)]. Quantitative polymerase chain reaction based on SYBR Green I was performed on a LightCycler^® 2.0 Real-Time PCR System (Roche Diagnostics GmbH, Mannheim, Germany) using the KAPA SYBR^® Fast Master Mix (2×) Universal Kit (Kapa Biosystems Inc., Wilmington, MA, USA) (Table 2) [21 (link)]. PCR reactions were performed in duplicate, in LightCycler^® glass capillaries, and contained 10 ng of each faecal DNA preparation (2 ng ΜL⁻¹), 10 μL of KAPA kit, 200 nM of each primer, 0.25 μL of Bovine Serum Albumin (BSA 20 mg mL⁻¹, New England Biolabs Inc, Hitchin, UK), and 3.95 μL ddH₂O. The thermal cycling conditions consisted of 3 min at 95 °C, followed by 45 cycles of 3 s at 95 °C, and then 20 s at 72 °C. Primer specificity was verified by performing a melting curve analysis. Microbial quantification was based on standard curves of genomic DNA from reference strains with the LightCycler^® software version 4.1 (Roche Diagnostics GmbH). Data are expressed as log₁₀ copies of 16S rRNA gene mL⁻¹ of sample [21 (link)].