Quantitative 16S rRNA Gene Analysis

Quantitative real-time PCR was performed to quantify the bacterial biomass in each gut sample. Amplification reactions were carried out in an ABI StepOne^TM Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) in a 10 μL mixture containing 1X BlasTaq^TM qPCR MasterMix (Applied Biological Materials Inc., Richmond, BC, Canada), 400 nM of each primer (341F and 515R [28 (link)]), and 1 μL of template DNA. The amplification conditions consisted of incubation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 60 s, and extension at 72 °C for 45 s. All samples were carried out in triplicate in optical 96-well plates along with the negative control and standard curve. Standard curve was created for absolute quantification of 16S rRNA gene using a plasmid containing the target gene fragment from Pseudomonas sp. DSM1650 10-fold diluted from 3.60 × 10⁷ to 3.60 × 10³ gene copy numbers μL⁻¹. Fluorescent light outputs were collected during each elongation step and analyzed with the ABI StepOne Real-Time PCR system SDS software v 2.3 (Applied Biosystems). Melting curves were generated after amplification by increasing the temperature of 0.5 °C every 30 s from 65 to 95 °C, in order to verify the absence of primer dimers or artifacts.