SVA Prevalence Estimation by RT-rtPCR

All PF samples were tested for the presence of SVA RNA by reverse-transcription real-time PCR (RT-rtPCR). RNA was extracted using a commercial extraction kit (Ambion MagMAX-96 viral RNA isolation kit; Life Technologies) and a magnetic particle processor (MagMAX Express-96 magnetic particle processor; Applied Biosystems), following the manufacturer’s guidelines. Although samples being assessed with RT-rtPCR are typically considered positive when the cycle threshold (Ct) values are 35.99 or lower and suspect when between 36 and 40 [16 (link)], we chose to classify any week as positive if at least one sample collected during that week had a Ct value under 40. Due to the decrease in sensitivity caused by aggregating multiple litters in one PF sample (due to a dilution effect) and since the prevalence of SVA-affected litters could be low (further decreasing sensitivity), suspect samples were treated as positives.