RNA Extraction and qRT-PCR for NDV Detection

RNA was extracted from the urine, fecal and throat samples by adding 60 μl sample to 90 μl Magnapure 96 external lysis buffer (6374913001, Roche Diagnostics, The Netherlands) as described before (Richard et al., 2020 (link)). Subsequently, the lysed sample was added to 60 μl Agencourt AMPure XP magnetic beads (A63880, Beckman Coulter, The Netherlands) and incubated 15 min at room temperature. Magnetic beads were washed three times with 70 % ethanol using the DynaMag-96 magnet (12027, Invitrogen, The Netherlands) and subsequently air-dried. RNA was eluted by 6 min of incubation in bidest H₂O. NDV-specific quantitative reverse transcription-PCR was performed using 5 μl RNA in an ABI PRISM 7000 Sequence Detection System using TaqMan Fast Virus 1-Step Master Mix (both from Thermo Fischer) in a total volume of 30 μl. The NDV-specific primers used were described by Wise et al. (2004) (link). The reverse transcriptase step was 5 min at 50 °C, followed by 95 °C for 20 s. Cycling consisted of 40 cycles of 3 s denaturation at 95 °C, 5 s annealing at 54 °C and 31 s extension at 60 °C.