2.4. Plasmid copy number determination by quantitative PCR

Templates for qPCR were obtained following the method described by Skulj, M. et al. [45]. The cells were subjected to incubation at 98 °C for 10 min, followed by centrifugation to collect the supernatant, which served as the template for qPCR. The qPCR primers were designed to target specific regions of both the EcN chromosome and the cryptic plasmid. Additionally, an AT-rich sequence (AATAAATCATAA) was added to the 5′ end of the primers, enhancing the qPCR fluorescence signal and improving the amplification efficiency [29,46]. We selected the recA and ldh genes on the EcN chromosome as reference genes, and their corresponding primers were recA-F/R and ldh-F/R, respectively. For targeting pMUT1 and pMUT2, we used T1-Rep-F/R and T2-Rep-F/R primers, respectively. The qPCR reactions were performed in a 50 μL mixture containing 25 μL of 2x SYBR Green®RPCR Master Mix (Vazyme), 200 nM of each primer (final concentration), and 4 μL of the sample. All qPCR reactions, including positive controls and negative controls (non-template control), were run on a QuantStudio3 instrument (Applied Biosystems). The universal cycling conditions for all reactions were as follows: an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 95 °C for 15 s, extension at 60 °C for 60 s, and a final extension at 95 °C for 15 s. The efficiency and specificity of the primers were evaluated by analyzing the standard curve and melting curve. The plasmid copy number was calculated using the comparative CT method (ΔΔC) based on the CT values obtained for the plasmid amplicons and the chromosome amplicons.