Flex purification system

RNA isolation was performed using a KingFisher Flex Purification System and a NucleoMag VET Kit (Macherey-Nagel, no. 744200.4) for the serum and swab samples as well as a NucleoMag RNA Kit (Macherey-Nagel, no. 744350.4) for the tissue samples, respectively, following the manufacturer’s instructions. 10 μL (2 × 10⁴ copies/μL) of enhanced green fluorescent protein (eGFP) RNA were added to each RNA isolation reaction as an internal control to verify the performance of the RNA isolation and real-time RT-PCR, as previously described (67 (link)), with modified primers and probe (Table S4).

Viral RNA was extracted from approximately 10 mg of spleen using the NucleoMag^® Vet kit (MACHEREY-NAGEL, Germany) on a KingFisher^™ Flex Purification System, according to the manufacturer’s instructions, and eluted with 100 μL of the supplied elution buffer. Complementary DNA (cDNA) was made using 2 μL qScript^™ Supermix (Quanta Biosciences, USA) and 8 μL RNA in a 10 μL reaction, according to the manufacturer’s instructions. Spleen samples (n = 191) were tested in duplicate on a Mic qPCR instrument (Bio Molecular Systems), using 1 μL template and PowerUp^™ SYBR^® Green Master Mix (Applied Biosystems, USA), with the primer concentration and cycling conditions as described previously [13 (link)]. Primers targeted the RNA dependent RNA polymerase gene within ORF1b [13 (link)]. Samples were considered positive if the amplification curve crossed the automatically defined threshold and the melting peak was between 85 °C and 86.5 °C. Samples were considered equivocal if only one of the duplicates was positive with the Cq value >33 or if both duplicates showed the correct melting peak, but the Cq was >37. Samples with equivocal results were retested in duplicate with 2 μL and 5 μL template, using a conventional PCR targeting a 321 bp conserved region in ORF1b [14 (link)].