S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen

Makadiya, Niraj; Brownlie, Robert; van den Hurk, Jan; Berube, Nathalie; Allan, Brenda; Gerdts, Volker; Zakhartchouk, Alexander

Piglet fecal swabs were collected in 0.5 ml DMEM on every day post-infection and stored at -80°C. RNeasy Plus Kit (Qiagen) was used for RNA isolation from the faecal swab samples. qRT-PCR was conducted in two steps: cDNA synthesis and PCR reactions. cDNA synthesis was performed with 1 μL (50 ng/μL) random hexamers, 1 μL of 10 mM dNTPs, and RNA in 13 μL volume and heated at 65°C for 5 min and chilled on ice followed by addition of 4 μL of 5X First-strand buffer, 1 μL of 0.1 M DTT and 1 μL of RNaseOUT (Thermo-Fisher Scientific) and 1 μL of SuperScript III enzyme (ThermoFisher Scientific) in final volume of 20 μL. The reaction conditions include 25°C for 5 min, 50°C for 60 min and 70°C for 15 min. The PCR reaction was performed in a total volume of 20 μL with 2 μl cDNA using Power SYBR green Master Mix (Qiagen); primers (5'-GCAACAACAGGTCCAGATCTC-3' and 5'-CTCCACG ACCCTGGTTATTTC-3') were present at 0.5 μM. PCR cycling conditions were: 95°C for 10 min and 41 cycles of 95°C for 15 s, 60°C for 1 min.