Tb green premix ex taq 2 tli rnase h plus 2

DNA was extracted from 0.2 g of each cecal contents sample using the QIAamp Fast DNA Stool Mini Kit for feces (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. The 16S rRNA gene and 11 tetracycline resistance genes (tetA, tetB, tetC, tetE, tetG, tetM, tetO, tetQ, tetT, tetW, and tetX) were detected and analyzed in this study. All qPCR assays were performed using the TB Green™ Premix Ex Taq™ kit (Takara Biotechnology Co. Ltd., Dalian, China) on the ABI StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). The qPCR mixture of the 11 genes were in a total volume of 10 μL, containing 5 μL TB Green Premix Ex Taq II (TliRNaseH Plus) (2×) (Takara Biotech, Co., Ltd., Dalian, China), 0.4 μL of each primer (primer sequences are referenced the study of Zhu et al. [39 (link)]), 0.2 μL ROX Reference Dye (50×), and 1 μL of DNA template, and the rest of the volume was made up of distillation-distillation H₂O. The thermal cycling steps for quantitative PCR amplification were as follows: (1) 95 °C for 30 s; (2) 95 °C for 5 s; (3) annealing temperature at 60 °C for 30 s, where steps (2) to (3) were repeated 40 times. The relative abundances of ARGs were calculated relative to 16S rRNA using the 2^−∆∆CT method [38 (link)].

RT-qPCR was performed on the CFX-96 TouchTM Real-Time PCR System (Bio-Rad, Hercules, CA, United States) using the TB Green Premix Ex Taq II (Tli RNase H Plus) (2×) (Takara, Japan). The internal control gene gyrB was used to normalize the expression of target genes, and the data were analyzed by using the 2^−ΔΔCt method. The relative expression of the target gene was normalized to that of S. aureus ATCC 29213. All RT-qPCR were performed in triplicate using 3 independent RNA samples. The RT-qPCR primers were listed in Supplementary Table S1.

The expression levels of P. aeruginosa QS circuit genes were evaluated by RT-qPCR, as described previously (55 (link)). P. aeruginosa cultures for bacteriophage-resistant strain TL3780-R (TL3780-R1 and TL3780-R2) and the parental strain TL3780 were incubated in fresh LB broth at 37°C under 180 rpm until reaching the logarithmic growth phase (OD₆₀₀ 0.5–0.6). Total RNA was extracted from planktonic bacteria using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. Purified RNA was reverse transcribed onto cDNA using the cDNA Synthesis Kit (TaKaRa, Tokyo, Japan) in accordance with the manufacturer’s instructions. The gene expression levels were measured by qRT-PCR using the TB Green Premix Ex Taq II (Tli RNase H Plus) (2×) (Takara) with specific primers listed in Table S3. rpsL was used as an internal control to normalize the data. The gene expression levels were calculated using the 2^−△△Ct method.

The total RNA of strains pET28b::rmpA FK2931 and pET28b::iucB::rmpA FK2931 were extracted. 500 ng RNA was then mixed with the reverse transcription system, and 10 μL of cDNA was obtained using a PrimeScript™ RT Kit (TaKaRa, Japan). Thereafter, by using a CFX-96 touch real-time PCR system, qPCR (Bio-Rad, CA, USA) was performed. After this step,100 ng cDNA, TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×) (TaKaRa), and the specific primers (rmpA(BamHI) F: 5’-CGGGATCCTACCGTGATTGATTGAATTTT-3’, rmpA(SacI) R: 5’-CGAGCTCTTACCTAAATACTTGGCATGAGC-3’) were added to each sample. The cycling conditions used were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The expression levels of gene rmpA were detected by RT-qPCR; 16S rRNA gene was used as the internal gene. In addition, compared with pET28b::rmpA FK2931, the target strain pET28b::iucB::rmpA FK2931 was quantified using the comparative threshold cycle 2^-ΔΔCt method. All experiments were repeated three times independently and average data was used for the calculation of relative expression levels.