Superscript 3 one step pcr system with platinum taq dna polymerase kit

According to the procedures outlined by Beck et al. [34 (link)], a quantitative real-time polymerase chain reaction (qRT-PCR) was employed to quantify the F. columnare adherent to the gill of the fish in all the treatment groups. DNA extraction was carried out using a DNeasy Blood and Tissue Kit from Qiagen per the manufacturer’s guidelines. The primers and FAM probe (Table 3), which were applied to target a portion of the F. columnare chondroitin AC lyase gene, were designed according to Panangala et al. [53 (link)]. A positive control consisted of a standard extracted template (1 × 10⁴ CFU/mL), and a negative control without extracted template was included in each run of the Q-PCR reactions. All samples were run in duplicate, and the reaction components and protocols of the qPCR were performed on a SimpliAmp Thermal Cycler (Thermo Fisher Scientific Inc., MA, USA) by using SuperScript^TM III One-Step PCR System with Platinum^® Taq DNA Polymerase kit (Invitrogen, Thermo Fisher Scientific, MA, USA). For normalization, the qPCR data were divided by the amount of template DNA put into each reaction, and the results are reported as CFU/ng of template DNA. Before counting the CFUs in a sample, a standard curve was created by plotting the qPCR data of bacteria serial dilutions against the CFU previously counted for each dilution.

According to the procedures outlined by Beck et al. [34 (link)], a quantitative real-time polymerase chain reaction (qRT-PCR) was employed to quantify the F. columnare adherent to the gill of the fish in all the treatment groups. DNA extraction was carried out using a DNeasy Blood and Tissue Kit from Qiagen per the manufacturer’s guidelines. The primers and FAM probe (Table 3), which were applied to target a portion of the F. columnare chondroitin AC lyase gene, were designed according to Panangala et al. [53 (link)]. A positive control consisted of a standard extracted template (1 × 10⁴ CFU/mL), and a negative control without extracted template was included in each run of the Q-PCR reactions. All samples were run in duplicate, and the reaction components and protocols of the qPCR were performed on a SimpliAmp Thermal Cycler (Thermo Fisher Scientific Inc., MA, USA) by using SuperScript^TM III One-Step PCR System with Platinum^® Taq DNA Polymerase kit (Invitrogen, Thermo Fisher Scientific, MA, USA). For normalization, the qPCR data were divided by the amount of template DNA put into each reaction, and the results are reported as CFU/ng of template DNA. Before counting the CFUs in a sample, a standard curve was created by plotting the qPCR data of bacteria serial dilutions against the CFU previously counted for each dilution.