Quantification of Avian IBV Genome Levels

Total ribonucleic acid (RNA) was extracted from the tissues and swabs using Trizol^® reagent (Invitrogen Canada Inc., Burlington, ON, Canada) following the manufacturer’s recommendations. RNA concentration was measured with a Nanodrop 1,000 spectrophotometer (ThermoScientific, Wilmington, DE, United States). To synthesize the cDNA from swabs and tissues, we used 1,000 and 2,000 ng of RNA, respectively, using random primers (High-Capacity Reverse Transcription Kit^™, Applied Biosystems, Invitrogen Canada Inc., Burlington, ON, Canada). The IBV genome load quantification was performed by real-time quantitative polymerase chain reaction (RT-qPCR) test using the CFX 96-c1000 Thermocycler (Bio-Rad Laboratories, Mississauga, ON, Canada). The RT-qPCR test was performed based on a SYBR^® Green Master Mix (Invitrogen, Burlington, ON, Canada). Each reaction was adjusted to 20 μL net volume, including 10 μL of SYBR Green master mix, 100 ng of cDNA per sample, and forward and reverse specific primers (0.5 μL for each). The primers used in this RT-qPCR assay targeted the nucleocapsid (N) gene of IBV as described previously (37 (link)). In order to quantify the absolute number of IBV genome copies, a standard curve was used using six ten-fold serial dilutions (10⁷–10²) of in-house prepared plasmids bearing the IBV-N gene (37 (link)).