2.6. Optimization of Reaction Conditions for Multiplex Real-Time PCR

For multiplex real-time PCR reaction systems, 10 μL of 2 × T5 Fast qPCR Mix was mixed with four primers (0.6 μL), probe (0.6 μL), template (1 μL), and nuclease-free water in a reaction system with a final volume of 20 μL. In order to investigate the ideal concentrations of primers and probes for this multiplex method, the multiplex reaction conditions were optimized using primers and probes of varying concentrations (10 μM). The method’s final primer and probe concentrations ranged from 300 nM to 500 nM and 100 nM to 300 nM, respectively. As templates, 2.18 × 10⁶ copies/μL of plasmid standards were used (Table 2).

The Cq values of PEDV, TGEV, PDCoV, and PoRVA detected by this multiplex real-time PCR assay with different probe and primer concentrations.