Quantifying Avian Coronavirus Genome Load

The quantification of IBV genome load in swab and tissue cDNA samples was performed with quantitative PCR (qPCR) assay using Fast SYBR^® Green Master Mix (Quntabio^®, Beverly, MA, USA). Each reaction volume incorporated 10 µL of SYBR Green master mix, 100 ng of interested cDNA sample, 0.5 µL 10 µM of forward primer, 0.5 µL 10 µM of reverse primer, and molecular biology-grade water to reach a final reaction volume of 20 µL. The qPCR assays were performed in a CFX 96-c1000 Thermocycler (Bio-Rad Laboratories, Mississauga, ON, Canada) with a thermal profile of 20 s of initial denaturation at 95 °C, 40 cycles of amplification with 3 s of denaturation at 95 °C, and 30 s of annealing at 60 °C. The forward and reverse primers used in this qPCR assay were 5′GACGGAGGACCTGATGGTAA-3′ and 5′CCCTTCTTCTGCTGATCCTG-3′, respectively, and they were targeted against the conserved IBV N gene [38 (link)].