Tb green premix ex taqtm 2 tli rnaseh plus reagent kit

As described in reference [23 (link)], the plasmid copy number (PCN) was determined using real-time quantitative PCR (qPCR). The copy number of the chromosomal single-copy atpF gene was used as an internal reference, and the copy number of the plasmid repA fragment was defined as the plasmid copy number. Bacterial genomic DNA was extracted using TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (TaKaRa, Japan) following the instruction manual, and qPCR was performed using the TB Green Premix Ex Taq^TM II (Tli RNaseH Plus) reagent kit (TaKaRa, Japan) on Applied Biosystems ViiA^TM 7 Dx (Life Technologies, USA). The thermal cycling parameters for the qPCR reaction were as follows: pre-denaturation at 95℃ for 7 min; denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, and 40 cycles. The PCN was calculated using the relative quantification formula 2^-ΔΔCT, with the reference gene being atpF. Primers used in this study were shown in Table S1.

Real-time quantitative reverse transcription PCR (qRT-PCR) was employed to detect the expression levels of antibiotic resistance-related genes, including bla_NDM-5, ompC, ompF, acrA, acrB, and tolC. Total bacterial RNA was extracted using the TRIzol-based method. The extracted RNA was reverse transcribed into cDNA using the PrimeScript^TM RT reagent Kit with gDNA Eraser (TaKaRa, Japan). qPCR was performed using the TB Green Premix Ex Taq^TM II (Tli RNaseH Plus) reagent kit (TaKaRa, Japan) on Applied Biosystems ViiA^TM 7 Dx (Life Technologies, USA). The thermal cycling parameters for the qPCR reaction were as follows: pre-denaturation at 95℃ for 7 min; denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, and 40 cycles. The relative expression level was calculated using the relative quantification formula 2^-ΔΔCT, with the reference gene being rpoD.