RT-qPCR for AvCoV genomic RNA was carried out using a Power SYBR® Green RNA-to-Ct™ 1-Step kit (Applied Biosystems) following the manufacturer’s instructions and primers described by Callison et al. (2006 (link)) targeting the 5ʹUTR, with the assays performed in triplicate. RT-qPCR of β-actin as an endogenous control was performed using a Power SYBR® Green RNA-to-Ct™ 1-Step kit (Applied Biosystems) and IDT Integrated DNA Technologies proprietary primers 5'ACAGAGCCTCGCCTTTG3'/5'CCTTGCACATGCCGGAG3', with the reactions performed in duplicate.
RT–qPCR was carried out in a 7500 Real-Time PCR system (Applied Biosystems) (48 °C/30 min; 95 °C/10 min; 45 cycles of 95 °C/15 s and 60 °C/40 s; and melting curve analysis). Absolute quantification (number of copies/µl of sample) of AvCoV was obtained by comparison with a tenfold dilution standard curve with a plasmid containing the corresponding 5′UTR sequence of AvCoV ranging from 1 × 103 to 1 × 109 copies per reaction (slope = − 3.536 and y-intercept = 43.665) and normalized to the β-actin Cq value of each sample.