Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Naïve Conventional Neonatal and Weaned Pigs

Thomas, Joseph T.; Chen, Qi; Gauger, Phillip C.; Giménez-Lirola, Luis G.; Sinha, Avanti; Harmon, Karen M.; Madson, Darin M.; Burrough, Eric R.; Magstadt, Drew R.; Salzbrenner, Holly M.; Welch, Michael W.; Yoon, Kyoung-Jin; Zimmerman, Jeffrey J.; Zhang, Jianqiang

The in vitro transcribed RNA was 10-fold serially diluted in nuclease-free water and used as standards in the PEDV N gene-based rRT-PCR to generate standard curves and quantify viral loads in test samples. Five μl of each RNA template was used in PCR setup in a 25μl total reaction using Path-ID™ Multiplex One-Step RT-PCR Kit (Thermo Fisher Scientific) as previously published [24, 25] . Amplification reactions were performed on an ABI 7500 Fast instrument (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with the following conditions: 1 cycle of 48°C for 10 min, 1 cycle of 95°C for 10 min, and 45 cycles of 95°C for 15 sec and 60°C for 45 sec. After generating 22 standard curves, the Ct values were averaged (means determined) and an equation of X = 10 (Ct-47.262)/-3.3969 , where X = genomic copies/ml, was developed to transform the Ct values into estimated genomic copies of PEDV RNA per ml in test samples under the PCR conditions of this study.