Quantitative RT-PCR Analysis of GBS

qRT-PCR was performed as previously described [19 (link),66 (link)]. Total RNA was isolated from GBS with TRIzol (Invitrogen Life Technologies, United States) after treatment with lysozyme for 30 min at 37℃. The quality of RNA was monitored by 1% electrophoresis and Nanodrop. Reverse transcription was conducted with 1 μg total RNA with EvoM-MLV RT kit with gDNA clean for qPCR (AG11705; Accurate Biology). And qRT-PCR was performed in a reaction volume of 10 μl including 5 μl 2×SYBR green premix pro Taq HS qPCR kit (AG11701; Accurate Biotechnology), 2.6 μl H₂O, 2 μl cDNA template, and 0.2 μl each of forward and reverse primers (10 mM) in 384-well plates. Primers used in this study was included in Supplementary Table S1. The reactions were set on a LightCycler 480 system (Roche, Germany). The cycling parameters were 95°C for 30 s, 40 cycles of 95°C for 10 s, and 60°C for 30 s. Fluorescence were measured at 72°C for 1 s during each cycle. At last, reaction was terminated at 95°C with a calefactive velocity of 5°C/s to obtain the melting curve. Data are shown as the relative mRNA expression compared with control group and standardized with the endogenous reference 16S rRNA gene. Statistic significance was calculated by 2^−ΔΔCt method [67 (link)].