Power sybr green pcr master mix

Quantification of AOB, NOB and total bacteria was performed using the primers CTO189R/CTO654R, NSR-1113F/NSR-1264R and 338F/518R. All the information of the primers was listed in S1 Table. The qPCR was carried out with 25 μl volume in eight-tubes-per-strip (Axygen) and 2× Power SYBR^® Green PCR Master Mix (Takara) was used. All PCR amplification used the same thermocycling steps as follows: 5 min at 95°C, followed by 40 cycles of 60 s at 95°C, 45 s at 57°C and 45 s at 72°C, and finally 10 min at 72°C [28 (link)]. The standard DNA was diluted to 1.47×10⁵−1.47×10¹⁰ (total bacteria 16S rDNA), 5.02×10⁴−5.02×10⁹ (AOB 16S rDNA), and 8.04×10⁴−8.04×10⁹ (NOB 16S rDNA) copies/μl, respectively. The reaction was carried out and monitored with an iCycler iQ^™ Real-Time PCR Detection System (Bio-Rad). The threshold cycle (Ct) values were measured after amplification and the copy numbers were calculated using the standard curve [29 (link)].

For reverse transcription-polymerase chain reaction (RT-PCR), first-strand cDNA was reverse-transcribed from the total RNA of Fp for RNA-seq using the Evo M-MLV RT Premix kit (AG11607, Accurate Biology, Hunan, China) according to the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) was performed using a CFX96 optical real-time detection system (Bio-Rad Laboratories Inc., Hercules, CA, United States). The 20 μL reactions contained 10 μL 2 × Power SYBR® Green PCR Master Mix (TaKaRa, Dalian, China), 0.4 μL of each 10 μM forward and reverse primers, 2.0 μL cDNA samples, and 7.2 μL sterile and DNA-free water. The PCR program was: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing at 54°C for 30 s, extension at 72°C for 20 s, for 40 cycles. Amplicon dissociation curves were recorded after cycle 40 by heating from 60 to 95°C with a ramp speed of 1.0°C per min. All reactions were performed with at least three technical replicates. TEF1α was used as the internal reference gene, and the relative expression was calculated using the 2^-ΔΔt method (Schmittgen and Livak, 2008 (link)). Twenty DEGs were selected, and primers were designed using Beacon Designer 7 and synthesized by Changsheng Biotechnology Co., Ltd. (Dingguo, Beijing, China). All primers are listed in Supplementary Table S1.

A quantitative polymerase chain reaction was performed to determine the total bacterial count. Amplification was performed using 2× Power SYBR Green PCR Master Mix (TAKARA BIO) according to the manufacturer's recommendations, using universal primers (Inubushi et al., 2010 ): forward primer (5′‐GTGSTGCAYGGYTGTCGTCA‐3′) and reverse primer (5′‐ACGTCRTCCMCACCTTCCTC‐3′). DNA was extracted from Porphyromonas gingivalis ATCC 33277 strain, adjusted for the bacterial count as the standard solution, and the bacterial count was calculated from the prepared calibration curve.